Quinoline derivatives having indoleamine-2,3-dioxygenase inhibitory activity

Abstract

The present application provides quinoline derivatives having indoleamine-2,3-dioxygenase inhibitory activity, in particular, the present application provides compounds of general formula (I) or pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, methods of preparation and uses in the manufacture of immunomodulatory and prophylactic and / or therapeutic agents for diseases associated with abnormal expression of IDO and / or abnormal tryptophan metabolism. The present application also relates to the combination of the quinoline derivatives with HDAC inhibitors and their use in the manufacture of anti-tumor agents.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of September 25, 2019, application number CN201910908030.5, and invention name “Quinoline derivatives with indoleamine-2,3-dioxygenase inhibitory activity”. Technical Field

[0002] This application relates to the field of medicine, specifically to quinoline derivatives with indoleamine 2,3-dioxygenase (IDO) inhibitory activity, or pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, preparation methods thereof, and their use in preparing drugs for immunomodulation and prevention and / or treatment of diseases associated with abnormal IDO expression and / or abnormal tryptophan metabolism. This application also relates to the combined use of the quinoline derivatives with epigenetic regulators and their use in preparing anti-tumor drugs. Background Art

[0003] Tryptophan (Trp) is an essential amino acid for the human body. A portion of the tryptophan obtained from the diet is used to synthesize proteins, niacin, and the neurotransmitter serotonin, while the remainder is metabolized primarily through the kynurenine pathway (Leklem JE, Am J Clin Nutr, 1971, 24(6): 659-672). IDO is a key enzyme involved in this metabolic pathway.

[0004] IDO is an intracellular heme-containing enzyme that was first discovered in the rabbit intestine in 1967 (Yamaoto S. et al., J Biol Chem, 1967, 242(22): 5260-5266). It is the only rate-limiting enzyme outside the liver that can catalyze the oxidative cleavage of the indole ring in the tryptophan molecule and its catabolism along the kynurenic acid pathway (MacKenzie, CR et al. Current Drug Metabolism, 2007, 8: 237-244).

[0005] The expression of IDO in cells and tissues is related to various inflammatory cytokines, especially IFN-γ. Other inflammatory cytokines such as IFN-α, IFN-β, TNF-α, and LPS can stimulate IDO expression at the transcriptional level (King NJ et al., The Int J Biochem Cell Biol, 2007 39(12):2167-2172). In addition, other immune active molecules such as prostaglandins, cell surface proteins cytotoxic T lymphocyte-associated antigen (CTLA-4), CD40, and Toll-like receptors can also regulate IDO expression.

[0006] It has been reported that there are two types of IDO, IDO-1 and IDO-2, and the main one with immunosuppressive function is IDO-1, while the role of IDO-2 in immunosuppression is not very clear. There are multiple factors in the mechanism of IDO-1 participating in immunosuppressive function. One of them is that high expression of IDO-1 causes local L-tryptophan depletion, so that the surrounding T lymphocytes can be sensed by GCN2 and other mechanisms, causing CD8 + Cytotoxic T cells to undergo cell cycle arrest or apoptosis. The second is that high expression of IDO-1 causes the increase of kynurenine (Kyn), a metabolite of tryptophan. After the formation of kynurenine, it can leave the cell and enter the extracellular matrix, and then enter the nearby lymphocytes by binding to the endogenous aromatic hydrocarbon receptor (AHR), thereby regulating CD8 + T cells and regulatory Treg cells, such as CD8 + The activity of cytotoxic T cells is inhibited, while the proliferation of regulatory Treg cells is induced and activated, thereby leading to the inhibition of immune activation function (Friberg M. et al., Int J Cancer, 2002, 101(2): 151-155). In addition, IDO-1 on antigen-presenting cells such as macrophages and dendritic cells (DC) can induce T cell immune tolerance to tumor antigens by inhibiting T cell proliferation (Terness P. et al., Blood, 2005, 105(6): 2480-2486).

[0007] IDO is closely related to the pathogenesis of many diseases, and has been confirmed as a target for major diseases such as cancer, Alzheimer's disease, depression, and cataract (CN101932325B, CN102579452B). IDO is closely related to nervous system diseases (Roy E.J. et al., Neurosci Lett, 2005, 387(2): 95-99). It is also involved in the occurrence of age-related nuclear cataract (Takikawa O. et al. Exp. Eye Res. 2001, 72: 271-277).

[0008] There are currently three IDO inhibitors in different clinical research stages, including: 1) Epacadostat of Incyte company, in clinical phase II, for the treatment of myelodysplastic syndrome, melanoma and female reproductive system cancer; 2) Indoximod of Newlink company, in clinical phase II, for the treatment of breast cancer, prostate cancer, malignant brain tumor, pancreatic cancer and melanoma; 3) GDC-0919 of Roche company, in clinical phase I, for the treatment of advanced solid tumors.

[0009] IDO inhibitors have good application prospects as drugs in the pharmaceutical industry. However, no suitable IDO inhibitors have been marketed to date. In order to achieve better disease treatment effects and better meet market demand, the development of a new generation of IDO inhibitors has great theoretical significance and application value.

[0010] Epigenetics is currently a hot topic in genetic research. It mainly includes DNA methylation, histone methylation, histone acetylation, phosphorylation, ubiquitination, and the activity of transcription factors involved in binding to histone modification sites. These epigenetic changes play an important role in gene transcription regulation. Histones are the basic building blocks of human chromosomes, and post-transcriptional modification of histones plays a decisive role in gene expression. Methylation of histone lysine or arginine sites, especially acetylation, is an important modification method. Numerous studies have shown that the imbalance of acetylation caused by the overexpression of histone deacetylase (HDAC) in cancer cells leads to the occurrence of tumors, and the inhibition of HDAC can effectively inhibit the formation of tumors.

[0011] However, there have been no studies or reports on the combined use of epigenetic regulators and IDO inhibitors in the treatment of tumors or tryptophan metabolism-related diseases. SUMMARY OF THE INVENTION

[0012] In one aspect, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0013] AXBYM

[0014] (I)

[0015] in,

[0016] A represents a 9-15 membered fused heteroaromatic ring containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur; optionally, the fused heteroaromatic ring is substituted by one or more substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl and hydroxy substituted C 1-6 alkyl;

[0017] X represents a covalent single bond, -C(R 1 R 2 )-、 -O-, -OC 1-6 Alkylene-, -NR 1 -, -S-, -S(O)-, or Among them, each R 1 and R 2 Each independently selected from hydrogen, halogen, hydroxy, amino, C1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl and hydroxy substituted C 1-6 alkyl;

[0018] B represents a 6-12 membered polycyclic aliphatic heterocycle containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur;

[0019] Y stands for Where D represents a covalent single bond or C 1-6 Alkylene, optionally, the C 1-6 The alkylene group is substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl and hydroxy substituted C 1-6 Alkyl; E represents a covalent single bond, C 1-6 Alkylene, C 2-6 Alkenylene, -NR 3 -or-C 1-6 Alkylene-NR 3 -, wherein the R 3 Selected from hydrogen, halogen, hydroxy, amino, C 1-6 Alkyl, halogenated C 1-6 Alkyl and hydroxy substituted C 1-6 alkyl;

[0020] M represents a 6-10 membered aromatic ring, a 3-7 membered aliphatic carbocyclic ring, a 5-10 membered monocyclic or polycyclic aromatic heterocyclic ring containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, a 3-7 membered saturated or partially unsaturated monocyclic alicyclic heterocyclic ring or a 6-12 membered polycyclic alicyclic heterocyclic ring containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur; optionally, the aromatic ring, carbocyclic ring, aromatic heterocyclic ring, aliphatic heterocyclic ring is substituted by one or more substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 2-6 Alkenyl, C 1-6 Alkoxy, C 1-6 Alkylamino, Halogenated C 1-6 Alkoxy, hydroxy substituted C 1-6 Alkoxy and C 1-6 Alkoxy-substituted C 2-6 Alkenyl.

[0021] In another aspect, the present application provides a pharmaceutical composition containing the compound disclosed in the present application or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0022] In another aspect, the present application provides a combination drug containing a first active ingredient and a second active ingredient, and optionally a pharmaceutically acceptable carrier or excipient; wherein the first active ingredient is selected from the compound disclosed in the present application or a pharmaceutically acceptable salt thereof, and other IDO inhibitors; and the second active ingredient is selected from epigenetic regulators.

[0023] In another aspect, the present application provides use of the disclosed compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a combination thereof in the preparation of an immunomodulator or a drug for preventing and / or treating diseases associated with abnormal IDO expression and / or abnormal tryptophan metabolism.

[0024] In another aspect, the present application provides the disclosed compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a combination drug for use in regulating individual immunity or preventing and / or treating diseases associated with abnormal IDO expression and / or abnormal tryptophan metabolism.

[0025] In another aspect, the present application provides a method for regulating immunity, comprising administering to a subject in need thereof an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a combination drug.

[0026] In another aspect, the present application provides a method for preventing and / or treating diseases related to abnormal IDO expression and / or abnormal tryptophan metabolism, comprising administering an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a combination drug thereof to a subject in need thereof.

[0027] In another aspect, the present application provides a method for preparing the disclosed compound of general formula (I), and the use of each intermediate involved in the preparation method for preparing the compound of general formula (I). Detailed Description of the Invention

[0029] Compound

[0030] In one aspect, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0031] AXBYM

[0032] (I)

[0033] in,

[0034] A represents a 9-15 membered fused heteroaromatic ring containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur; optionally, the fused heteroaromatic ring is substituted by one or more substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C1-6 alkyl and hydroxy-substituted C 1-6 alkyl;

[0035] X represents a covalent single bond, -C(R 1 R 2 )-, -O-, -O-C 1-6 alkylene-, -NR 1 -, -S-, -S(O)- or wherein each R 1 and R 2 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkyl and hydroxy-substituted C 1-6 alkyl;

[0036] B represents a 6-12 membered polycyclic aliphatic heterocycle containing 1-3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur;

[0037] Y represents wherein D represents a covalent single bond or C 1-6 alkylene, optionally, said C 1-6 alkylene is substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, haloC 1-6 alkyl and hydroxy-substituted C 1-6 alkyl; E represents a covalent single bond, C 1-6 alkylene, C 2-6 alkenylene, -NR 3 - or -C 1-6 alkylene-NR 3 - wherein said R 3 is selected from the group consisting of hydrogen, halogen, hydroxy, amino, C 1-6 alkyl, haloC 1-6 alkyl and hydroxy-substituted C 1-6 alkyl;

[0038] M represents a 6-10 membered aromatic ring, a 3-7 membered aliphatic carbon ring, a 5-10 membered monocyclic or polycyclic aromatic heterocycle containing 1-4 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, a 3-7 membered saturated or partially unsaturated monocyclic aliphatic heterocycle containing 1-5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, or a 6-12 membered polycyclic aliphatic heterocycle; optionally, said aromatic ring, carbon ring, aromatic heterocycle, aliphatic heterocycle is substituted with one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 1-6 alkylamino, Halogenated C 1-6 Alkoxy, hydroxy substituted C 1-6 Alkoxy and C 1-6 Alkoxy-substituted C 2-6 Alkenyl.

[0039] In some preferred embodiments, A represents a 9-10 membered fused heteroaromatic ring containing 1-2 heteroatoms selected from nitrogen, oxygen and sulfur; optionally, the fused heteroaromatic ring is substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 In some preferred embodiments, A represents a 9-10 membered fused heteroaromatic ring containing 1-2 nitrogen atoms; optionally, the fused heteroaromatic ring is substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, amino, C 1-2 Alkyl, C 1-2 Alkoxy, halogenated C 1-2 Alkyl and hydroxy substituted C 1-2 In some preferred embodiments, A is selected from the following groups optionally substituted with one or more halogens: benzimidazolyl, imidazopyridinyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, and naphthyridinyl. In some preferred embodiments, A is 6-fluoroquinolyl.

[0040] In some preferred embodiments, X represents a covalent single bond, -C(R 1 R 2 )-、 -O-, -OC 1-4 Alkylene-, -NR 1 -, -S-, -S(O)-, or Among them, each R 1 and R 2 Each independently selected from hydrogen, halogen, hydroxy, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 In some preferred embodiments, X represents a covalent single bond, -O-, -OC 1-2 Alkylene- or -NR 1 -; each R 1 and R 2 are each independently selected from hydrogen and C 1-2Alkyl. In some preferred embodiments, X represents a single covalent bond, -O-, -OCH2-, -NH-, or -N(CH3)-. In some preferred embodiments, X represents -O-, -NH-, or -N(CH3)-. In some preferred embodiments, X represents -O- or -NH-. In some preferred embodiments, X represents -O-. In some embodiments, X represents a single covalent bond. In some embodiments, X represents -N(CH3)-.

[0041] In some preferred embodiments, B represents a 6-9 membered polycyclic alicyclic heterocycle containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur; preferably, at least one of the heteroatoms is nitrogen. In some preferred embodiments, B represents a 6-9 membered bicyclic alicyclic heterocycle containing 1-3 heteroatoms of nitrogen, oxygen and sulfur. In some preferred embodiments, at least one of the heteroatoms is nitrogen. In some preferred embodiments, B represents a 6-9 membered spiroheterocycle or a 6-9 membered heterocycle containing 1-3 nitrogen atoms. In some preferred embodiments, B is connected to Y via a nitrogen atom on its ring. In some preferred embodiments, B is selected from In some preferred embodiments, B is selected from In some embodiments, B is In some embodiments, B is In some embodiments, B is

[0042] In some preferred embodiments, Y represents Where D represents a covalent single bond or C 1-4 Alkylene, optionally, the C 1-4 The alkylene group is substituted by one or more substituents selected from the group consisting of halogen, hydroxy, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 Alkyl; E represents a covalent single bond, C 1-4 Alkylene, C 2-4 Alkenylene, -NR 3 -or-C 1-4 Alkylene-NR 3 -, wherein the R 3 Selected from hydrogen, halogen, hydroxy, amino, C 1-4 Alkyl, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 In some preferred embodiments, Y represents Where D represents a covalent single bond or C 1-2 Alkylene, optionally, Y represents Where D represents a covalent single bond or C1-2 Alkylene, optionally, the C 1-2 Alkylene is substituted by one or more hydroxyl groups 1-2 Alkyl; E represents a covalent single bond, vinylidene, -NH- or -C 1-2 In some preferred embodiments, Y represents -C(O)-, In some embodiments, Y represents -C(O)-, In some embodiments, Y represents In some embodiments, Y represents

[0043] In some preferred embodiments, M represents a 6-10 membered aromatic ring, a 3-7 membered aliphatic carbocyclic ring, a 5-6 membered monocyclic aromatic heterocycle containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, or an 8-10 membered bicyclic aromatic heterocycle, or a 3-6 membered monocyclic alicyclic heterocycle containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur, or a 7-10 membered bicyclic alicyclic heterocycle; optionally, the aromatic ring, carbocyclic ring, aromatic heterocycle, or alicyclic heterocycle is substituted by one or more substituents selected from the following: halogen, hydroxyl, amino, C 1-4 Alkyl, C 2-4 Alkenyl, C 1-4 Alkoxy, C 1-4 Alkylamino, Halogenated C 1-4 Alkoxy, hydroxy substituted C 1-4 Alkoxy and C 1-4 Alkoxy-substituted C 2-4 In some preferred embodiments, M represents a benzene ring, a 3-7 membered aliphatic carbon ring, a 5-6 membered monocyclic aromatic heterocycle containing 1-2 heteroatoms selected from nitrogen, oxygen and sulfur, or an 8-10 membered bicyclic aromatic heterocycle, or a 3-6 membered monocyclic alicyclic heterocycle containing 1-2 heteroatoms selected from nitrogen, oxygen and sulfur; optionally, the benzene ring, carbon ring, 5-6 membered monocyclic aromatic heterocycle, 8-10 membered bicyclic aromatic heterocycle, or 3-6 membered monocyclic alicyclic heterocycle is substituted by one or more substituents selected from the following: halogen, hydroxyl, amino, C 1-2 Alkyl, vinyl, C 1-2 Alkoxy, C 1-2 Alkylamino, Halogenated C 1-2 Alkoxy, hydroxy substituted C 1-2 Alkoxy and C 1-2 Alkoxy-substituted C 2-4 In some preferred embodiments, M represents a benzene ring, a 3-7 membered aliphatic carbocyclic ring, a 5-6 membered monocyclic aromatic heterocycle containing 1-2 heteroatoms selected from nitrogen and oxygen, or an 8-10 membered bicyclic aromatic heterocycle; optionally, the benzene ring, carbocyclic ring, 5-6 membered monocyclic aromatic heterocycle, or 8-10 membered bicyclic aromatic heterocycle is substituted by one or more substituents selected from the following: halogen, C1-2 Alkoxy, and C 1-2 Alkoxy-substituted C 2-4 In some preferred embodiments, M represents a benzene ring, a cyclohexane ring, a pyridine ring, a 2,3-dihydrobenzofuran ring, a benzo[1,3]dioxole ring, or a 2,3-dihydrobenzo[1,4]dioxane ring; optionally, the benzene ring is substituted with one or more substituents selected from the group consisting of fluorine, chlorine, methoxy, dimethylamino, and methoxy-substituted propenyl. In some embodiments, M represents a benzene ring, a halogenated benzene ring (e.g., a chlorinated benzene ring, a fluorinated benzene ring), or a methoxy-substituted benzene ring. In some embodiments, M represents a halogenated benzene ring. In some embodiments, M represents a 4-chlorobenzene ring.

[0044] In some preferred embodiments, B represents a 6-9 membered bicyclic alicyclic heterocycle containing 1-3 nitrogen, oxygen and sulfur heteroatoms;

[0045] Y stands for Where D represents a covalent single bond or C 1-4 Alkylene, optionally, the C 1-4 Alkylene is substituted by one or more hydroxyl groups 1-4 Alkyl; E represents a covalent single bond, vinylidene, -NH- or -C 1-4 Alkylene-NH-; E represents a covalent single bond, C 1-4 Alkylene, C 2-4 Alkenylene, -NR 3 -or-C 1-4 Alkylene-NR 3 -, wherein the R 3 Selected from hydrogen, halogen, hydroxy, amino, C 1-4 Alkyl, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 alkyl.

[0046] In some preferred embodiments, B represents a 6-9 membered spiroheterocyclic ring or a 6-9 membered heterocyclic ring containing 1-3 nitrogen atoms;

[0047] Y stands for Where D represents a covalent single bond or C 1-2 Alkylene, optionally, the C 1-2 Alkylene is substituted by one or more hydroxyl groups 1-2 Alkyl; E represents a covalent single bond, vinylidene, -NH- or -C 1-2 Alkylene-NH-; and, B is connected to Y through the nitrogen atom on its ring.

[0048] In some preferred embodiments, -BY- is selected from In some preferred embodiments, -BY- is selected from

[0049] In some preferred embodiments, X represents a covalent single bond, -C(R 1 R 2 )-、 -O-, -OC 1-4 Alkylene-, -NR 1 -, -S-, -S(O)-, or Among them, each R 1 and R 2 Each independently selected from hydrogen, halogen, hydroxy, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 alkyl;

[0050] B represents a 6-9 membered bicyclic aliphatic heterocycle containing 1-3 nitrogen, oxygen and sulfur heteroatoms;

[0051] Y stands for Where D represents a covalent single bond or C 1-4 Alkylene, optionally, the C 1-4 Alkylene is substituted by one or more hydroxyl groups 1-4 Alkyl; E represents a covalent single bond, vinylidene, -NH- or -C 1-4 Alkylene-NH-; E represents a covalent single bond, C 1-4 Alkylene, C 2-4 Alkenylene, -NR 3 -or-C 1-4 Alkylene-NR 3 -, wherein the R 3 Selected from hydrogen, halogen, hydroxy, amino, C 1-4 Alkyl, halogenated C 1-4 Alkyl and hydroxy substituted C 1-4 alkyl.

[0052] In some preferred embodiments, X represents a covalent single bond, -O-, -OC 1-2 Alkylene- or -NR 1 -; each R 1 and R 2 are each independently selected from hydrogen and C 1-2 alkyl;

[0053] B represents a 6-9 membered spiro heterocycle or a 6-9 membered heterocycle containing 1-3 nitrogen atoms;

[0054] Y stands for Where D represents a covalent single bond or C 1-2 Alkylene, optionally, the C 1-2 Alkylene is substituted by one or more hydroxyl groups 1-2 Alkyl; E represents a covalent single bond, vinylidene, -NH- or -C 1-2 Alkylene-NH-; and, B is connected to Y through the nitrogen atom on its ring.

[0055] In some preferred embodiments, X represents a covalent single bond, -O-, -OCH2-, -NH- or -N(CH3)-;

[0056] -BY-Selected

[0057] In some preferred embodiments, the compound described herein is selected from:

[0058]

[0059]

[0060] In this document, for the divalent structure defined as "X", "B", "Y", "D" or "E" in the compound of general formula (I), both ends of the divalent structure may be connected to the adjacent structure, preferably connected to the adjacent structure according to the writing format, that is, the left end is connected to the left structure, and the right end is connected to the right structure. In some embodiments, when E is -C 1-6 Alkylene-NR 3 -, it can pass the C on the left 1-6 The alkylene group is connected to the carbonyl group in Y, or it can be connected to the right side of the -NR 3 - is connected to the carbonyl group in Y.

[0061] Pharmaceutical composition

[0062] When administered as a drug, the compound can be administered in the form of a pharmaceutical composition. Therefore, in another aspect, the present application provides a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0063] The term "composition" is used to refer to a product containing a compound disclosed in this application or a pharmaceutically acceptable salt thereof as the specific active ingredient, as well as any other product that is in combination with the active ingredient directly or indirectly.

[0064] Usually, the pharmaceutical composition contains at least one pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients in the formula and is harmless to the subject. The carrier described here refers to a substance used to improve the selectivity, effectiveness and / or safety of the drug during delivery. The carrier is mainly used to control drug release and can also be used to improve the pharmacokinetic properties of the drug, especially bioavailability. The excipient refers to other substances in the pharmaceutical preparation other than the active ingredient, which are mainly used for long-term stability, filling solid preparations (therefore, it is often used to specifically refer to "fillers") or enhancing product efficacy (for example, promoting absorption, reducing viscosity or increasing solubility, etc.).

[0065] In some embodiments, the pharmaceutical composition further contains other IDO inhibitors. In some preferred embodiments, the IDO inhibitor may be a tryptophan analogue (e.g., Indoximod) or an IDO1 inhibitor (e.g., Epacadostat, NLG919). In some preferred embodiments, the pharmaceutical composition further contains an epigenetic regulator. In some preferred embodiments, the epigenetic regulator is selected from DNA methylase inhibitors (e.g., azacitidine (5-Aza), decitabine (Decitabine)), histone methylase inhibitors (e.g., EZH2 inhibitors), histone demethylase inhibitors (e.g., LSD inhibitors), histone deacetylase (HDAC) inhibitors. In some preferred embodiments, the HDAC inhibitor is selected from benzamide HDAC inhibitors (e.g., Chidamide), hydroxamic acid HDAC inhibitors (e.g., Vorinostat), cyclic peptide HDAC inhibitors (e.g., Romidepsin), and short-chain fatty acid HDAC inhibitors (e.g., valproic acid).

[0066] In some preferred embodiments, the pharmaceutical composition contains 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient; optionally, it further contains one or more other therapeutic agents. In some preferred embodiments, the other therapeutic agent may be another IDO inhibitor. In some preferred embodiments, the IDO inhibitor may be a tryptophan analog (e.g., Indoximod) or an IDO1 inhibitor (e.g., Epacadostat, NLG919). In some preferred embodiments, the other therapeutic agent may be an epigenetic regulator. In some preferred embodiments, the epigenetic regulator is selected from DNA methylase inhibitors (e.g., azacitidine (5-Aza), decitabine (Decitabine)), histone methylase inhibitors (e.g., EZH2 inhibitors), histone demethylase inhibitors (e.g., LSD inhibitors), histone deacetylase (HDAC) inhibitors. In some preferred embodiments, the HDAC inhibitor is selected from benzamide HDAC inhibitors (e.g., Chidamide), hydroxamic acid HDAC inhibitors (e.g., Vorinostat), cyclic peptide HDAC inhibitors (e.g., Romidepsin), and short-chain fatty acid HDAC inhibitors (e.g., valproic acid). In some preferred embodiments, the pharmaceutical composition contains 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one or a pharmaceutically acceptable salt thereof and chidamide and at least one pharmaceutically acceptable carrier or excipient.

[0067] In some preferred embodiments, the pharmaceutical composition contains N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide, N-(benzo[d][1,3]dioxolan-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide, 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3- In some preferred embodiments, the present invention relates to a pharmaceutical composition comprising: ... In some preferred embodiments, the epigenetic regulator is selected from DNA methylase inhibitors (e.g., azacitidine (5-Aza), decitabine (Decitabine)), histone methylase inhibitors (e.g., EZH2 inhibitors), histone demethylase inhibitors (e.g., LSD inhibitors), histone deacetylase (HDAC) inhibitors. In some preferred embodiments, the HDAC inhibitor is selected from benzamide HDAC inhibitors (e.g., Chidamide), hydroxamic acid HDAC inhibitors (e.g., Vorinostat), cyclic peptide HDAC inhibitors (e.g., Romidepsin), and short-chain fatty acid HDAC inhibitors (e.g., valproic acid).In some preferred embodiments, the pharmaceutical composition contains N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide, N-(benzo[d][1,3]dioxolan-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide, 2-(6-((6-fluoroquinolin-4-yl)amino)-3- Azabicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide or 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one or a pharmaceutically acceptable salt thereof, as well as chidamide and at least one pharmaceutically acceptable carrier or excipient.

[0068] Combination therapy

[0069] Epigenetics is a hot topic in genetics research, which mainly includes DNA methylation, histone methylation, histone acetylation, phosphorylation, ubiquitination, and transcription factor activity involved in binding to histone modification sites, etc. These epigenetic changes play an important role in gene transcription regulation. DNA methylation mainly occurs at CpG dinucleotide sites in the genome, which is catalyzed by DNA methyltransferase. Generally, DNA methylation is negatively correlated with gene expression. Studies have shown that abnormal DNA methylation can participate in tumor formation by affecting chromatin structure and the expression of oncogenes and tumor suppressor genes. The commonly used DNA methyltransferase inhibitors in clinic include azacitidine (5-Aza), decitabine (Decitabine), etc. Histone is the basic unit of human chromosome, and post-transcriptional modification of histone plays a decisive role in gene expression. Methylation of histone lysine or arginine sites, especially acetylation, is an important modification method. A large number of studies have shown that the imbalance of acetylation caused by the overexpression of HDAC in cancer cells leads to the occurrence of tumors, and the generation of tumors can be well inhibited by inhibiting HDAC. At present, the histone methylation enzyme inhibitors entering the clinical trial stage include EZH2 inhibitors, LSD inhibitors, etc. HDAC inhibitors include benzamide HDAC inhibitors (such as chidamide), hydroxamic acid HDAC inhibitors (such as vorinostat), cyclic peptide HDAC inhibitors (such as romidepsin), and short-chain fatty acid HDAC inhibitors (such as valproic acid). In addition, transcription factors binding to histone modification sites, such as BET protein family members containing bromodomain, also participate in cyclin expression, chromatin shaping, and even directly promote the abnormal expression of oncogenes such as Myc to induce tumor occurrence. Some BET inhibitors have entered the clinical research stage.

[0070] The present inventors have surprisingly found that in a tumor model constructed by inoculating CT-26 colon cancer cells into Balb / c mice, the compound having IDO inhibitory activity and the HDAC inhibitor show excellent synergistic effect, and the anti-tumor activity is significantly better than that of single-target IDO inhibitor or HDAC inhibitor. Therefore, another aspect of the present application provides a combination drug, which contains a first active ingredient and a second active ingredient, and optionally a pharmaceutically acceptable carrier or excipient; wherein the first active ingredient is selected from the compounds disclosed in the present application or pharmaceutically acceptable salts thereof, and other IDO inhibitors; the second active ingredient is selected from epigenetic regulators.

[0071] In some preferred embodiments, the IDO inhibitor is selected from tryptophan analogs (e.g., Indoximod) and IDO1 inhibitors (e.g., Epacadostat, NLG919). In some preferred embodiments, the epigenetic regulator is selected from DNA methylase inhibitors (e.g., azacitidine (5-Aza), decitabine (Decitabine)), histone methylase inhibitors (e.g., EZH2 inhibitors), histone demethylase inhibitors (e.g., LSD inhibitors) and HDAC inhibitors. In some preferred embodiments, the HDAC inhibitor is selected from benzamide HDAC inhibitors (e.g., Chidamide), hydroxamic acid HDAC inhibitors (e.g., Vorinostat), cyclic peptide HDAC inhibitors (e.g., Romidepsin) and short-chain fatty acid HDAC inhibitors (e.g., valproic acid). In some preferred embodiments, the first active ingredient and the second active ingredient are in the same formulation unit. In some preferred embodiments, the first active ingredient and the second active ingredient are in different formulation units. In some preferred embodiments, the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially. In some preferred embodiments, the first active ingredient is N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide, or a pharmaceutically acceptable salt thereof, and the second active ingredient is chidamide.

[0072] In some preferred embodiments, the first active ingredient is 2-(benzo[d][l,3]dioxol-5-ylamino)-l-(4-(2-(6-fluoro-5H-imidazo[5,l-a]isoindol-5-yl)-l- hydroxyethyl)piperidin-l-yl)ethyl-l-one or a pharmaceutically acceptable salt thereof; and the second active ingredient is selected from the group consisting of epigenetic modulators. In some preferred embodiments, the epigenetic modulator is selected from the group consisting of DNA methyltransferase inhibitors (e.g. azacitidine (5-Aza), decitabine), histone methyltransferase inhibitors (e.g. EZH2 inhibitors), histone demethylase inhibitors (e.g. LSD inhibitors), histone deacetylase (HDAC) inhibitors. In some preferred embodiments, the HDAC inhibitor is selected from the group consisting of benzamide HDAC inhibitors (e.g. chidamide), hydroxamic acid HDAC inhibitors (e.g. vorinostat), cyclic peptide HDAC inhibitors (e.g. romidepsin), and short-chain fatty acid HDAC inhibitors (e.g. valproic acid). In some preferred embodiments, the first active ingredient is 2-(benzo[d][l,3]dioxol-5-ylamino)-l-(4-(2-(6-fluoro-5H-imidazo[5,l-a]isoindol-5-yl)-l- hydroxyethyl)piperidin-l-yl)ethyl-l-one or a pharmaceutically acceptable salt thereof; and the second active ingredient is chidamide.

[0073] In some preferred embodiments, the first active ingredient is selected from N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide, N-(benzo[d][1,3]dioxolane-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide, 2-(6-((6-fluoroquinolin-4-yl)amino )-3-azabicyclo[3.1.0]hexane-3-yl)-N-(4-methoxyphenyl)propionamide and 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one or a pharmaceutically acceptable salt thereof; the second active ingredient is selected from epigenetic regulators. In some preferred embodiments, the epigenetic regulator is selected from DNA methylase inhibitors (such as azacitidine (5-Aza), decitabine), histone methylase inhibitors (such as EZH2 inhibitors), histone demethylase inhibitors (such as LSD inhibitors), and histone deacetylase (HDAC) inhibitors. In some preferred embodiments, the HDAC inhibitor is selected from benzamide HDAC inhibitors (e.g., chidamide), hydroxamic acid HDAC inhibitors (e.g., vorinostat), cyclic peptide HDAC inhibitors (e.g., romidepsin), and short-chain fatty acid HDAC inhibitors (e.g., valproic acid). In some preferred embodiments, the first active ingredient is 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one or a pharmaceutically acceptable salt thereof; the second active ingredient is chidamide.

[0074] use

[0075] As previously mentioned, IDO1 has immunosuppressive functions and is closely linked to the pathogenesis of numerous diseases. For example, it is a target for major illnesses such as cancer, Alzheimer's disease, depression, and cataracts. Furthermore, it is implicated in the development of age-related nuclear cataracts. In the activity assay, the compounds disclosed herein demonstrated significant inhibitory activity against hIDO1.

[0076] Therefore, in another aspect, the present application provides the use of the compound or a pharmaceutically acceptable salt thereof, pharmaceutical composition, or combination thereof in the preparation of an immunomodulator or a medicament for preventing and / or treating a disease associated with abnormal IDO expression and / or abnormal tryptophan metabolism. In some preferred embodiments, the disease is selected from tumors, autoimmune diseases, cataracts, Alzheimer's disease, depressive disorders, and anxiety disorders.

[0077] The present application also provides the compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a combination thereof for use in regulating individual immunity or preventing and / or treating diseases associated with abnormal IDO expression and / or abnormal tryptophan metabolism. In some preferred embodiments, the disease is selected from tumors, autoimmune diseases, cataracts, Alzheimer's disease, depressive disorders, and anxiety disorders.

[0078] The present application also provides a method for regulating immunity, comprising administering an effective amount of the compound disclosed in the present application or a pharmaceutically acceptable salt thereof, a pharmaceutical composition or a combination drug to a subject in need thereof.

[0079] The present application also provides a method for preventing and / or treating a disease associated with abnormal IDO expression and / or abnormal tryptophan metabolism, comprising administering to a subject in need thereof an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, pharmaceutical composition, or combination thereof. In some preferred embodiments, the disease is selected from the group consisting of tumors, autoimmune diseases, cataracts, Alzheimer's disease, depressive disorders, and anxiety disorders.

[0080] As used herein, the term "subject" or "individual" refers to an animal, including but not limited to a primate (e.g., a human), a cow, a pig, a sheep, a goat, a horse, a dog, a cat, a rabbit, a rat, or a mouse. "Individual" and "patient" are used interchangeably herein, e.g., with reference to a mammalian subject, e.g., a human subject. In some embodiments, the subject is a human.

[0081] As used herein, the term "treating" is intended to include alleviating or eliminating the condition, disorder or disease, or one or more symptoms associated with the condition, disorder or disease; or alleviating or eliminating the cause of the condition, disorder or disease itself.

[0082] As used herein, the term "prevent," "prevent," "prevent," "prevent," "prevent," "preventing," "prevent ...

[0083] The term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutically effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the method of administration of the drug, and any other concurrently administered treatments.

[0084] Preparation method

[0085] In another aspect, the present application provides a method for preparing the compound of general formula (I), which is selected from the following methods:

[0086] Method 1:

[0087] When Y is D is a covalent single bond, E is -NR 3 -, the compound of general formula (I) can be prepared by the following method: in an organic solvent, in the presence of a base, reacting a compound of formula I-1, a compound of formula I-2 and a urea reagent to generate a compound of general formula (I),

[0088]

[0089] Method 2:

[0090] When Y is D is a covalent single bond, E is a covalent single bond, C 1-6 Alkylene, C 2-6 Alkenylene or -C 1-6 Alkylene-NR 3 -, the compound of general formula (I) can be prepared by the following method: a compound of formula I-1 and a compound of formula I-3 are subjected to a coupling reaction to generate a compound of general formula (I);

[0091]

[0092] Method 3:

[0093] When Y is D is substituted or unsubstituted C 1-6 Alkylene, E is -NR 3 -, the compound of general formula (I) can be prepared by the following method: a compound of formula I-4 and a compound of formula I-2 are subjected to coupling reaction to generate a compound of general formula (I),

[0094]

[0095] wherein A, X, B, R 3 and M are as defined above.

[0096] In some preferred embodiments, B represents a 6-12 membered heteropolycycloalkyl group containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur, and at least one of the heteroatoms is nitrogen, and B is connected to Y through a nitrogen atom on the ring of B.

[0097] In some preferred embodiments, in Method 1(1), the organic solvent can be dichloromethane, THF, toluene, ethyl acetate or benzene, etc. In some preferred embodiments, in Method 1(1), the base is triethylamine, pyridine, DIPEA or NaOH. In some preferred embodiments, in Method 1(1), the urea-forming reagent is selected from isocyanate, triphosgene, phosgene, diphosgene, chloroformamide, carbonyl diimidazole (CDI) and potassium isocyanate.

[0098] In some preferred embodiments, in Method 2 and / or Method 3, the coupling reaction is catalyzed by a peptide condensing agent, such as 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N,N'-dicyclohexyl carbodiimide (DCC), N,N'-carbonyl diimidazole (CDI), O-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), etc. In some preferred embodiments, in Method 2 and / or Method 3, the coupling reaction temperature is 0-60°C. In some preferred embodiments, in Method 2 and / or Method 3, the coupling reaction time is 2-72 hours. In some preferred embodiments, in Method 2 and / or Method 3, the solvent used in the coupling reaction is selected from benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform and N,N'-dimethylformamide, etc. In some preferred embodiments, in Method 1(2) and / or Method 2, a base such as sodium hydroxide, triethylamine, DMAP or pyridine, etc. can also be added to the coupling reaction if necessary.

[0099] The compound of general formula (I) can be purified by common separation methods, such as extraction, recrystallization, column chromatography, etc.

[0100] Preparation of compound of formula I-1

[0101] In addition, the present application further provides a preparation method of the compound of formula I-1, which comprises steps (a)-(b):

[0102] (a) under the action of a base, compound 1 and compound 2 undergo a nucleophilic substitution reaction to obtain compound 3:

[0103]

[0104] (b) Under the action of acid, compound 3 is deprotected to obtain formula I-1:

[0105]

[0106] wherein A, X and B are as defined above.

[0107] In some preferred embodiments, the nucleophilic substitution reaction in step (a) uses a base such as KHMDS, NaH, NaOH, Na2CO3, or K2CO3 as a deacidifying agent. In some preferred embodiments, the reaction temperature in step (a) is 25-140°C. In some preferred embodiments, the reaction time in step (a) is 2-72 hours. In some preferred embodiments, the solvent used in the nucleophilic substitution reaction in step (a) is selected from water, methanol, ethanol, acetonitrile, benzene, xylene, acetone, N,N'-dimethylformamide, and DMSO.

[0108] In some preferred embodiments, the acid in step (b) is selected from trifluoroacetic acid and hydrogen chloride solution. In some preferred embodiments, the base in step (b) is NaOH. In some preferred embodiments, the reaction temperature in step (b) is 25-140°C. In some preferred embodiments, the reaction time in step (b) is 2-72 hours. In some preferred embodiments, the solvent used in step (b) is selected from water, methanol, ethanol, acetonitrile, benzene, xylene, acetone, N,N'-dimethylformamide, and DMSO.

[0109] Preparation of compound of formula I-4

[0110] In addition, the present application further provides a method for preparing a compound of formula I-4, comprising steps (c)-(d):

[0111] (c) a compound of formula I-1 reacts with a halogenated carboxylic acid ester 4 (e.g., chloroformate, 2-bromoacetate, 2-bromopropionate, 1-hydroxy-2-bromopropionate) in the presence of a base to produce a compound 5 through a nucleophilic substitution reaction;

[0112]

[0113] (d) Compound 5 is hydrolyzed under alkaline conditions to obtain a compound of formula I-4;

[0114]

[0115] Among them, R 4 Representative C 1-6 Alkyl, A, X, B and D are as defined above.

[0116] In particular, when the halogenated carboxylate in step (c) is a 2-halogenated propionic acid ester, the compound of formula I-4 has the structure shown in formula F:

[0117]

[0118] Accordingly, the present application further provides a method for preparing the compound of formula F as described above, which comprises steps (e)-(f):

[0119] (e) a nucleophilic substitution reaction of the compound of formula I-1 with a 2-halogenated propionic acid ester (e.g. ethyl 2-bromopropionate) in the presence of a base to obtain compound 6;

[0120]

[0121] (f) a hydrolysis reaction of compound 4 under basic conditions to obtain the compound of formula F;

[0122]

[0123] In some preferred embodiments, the nucleophilic substitution reaction in step (c) and / or (e) is carried out with a base selected from NaOH, Na2CO3, K2CO3, and the like as a deacidifying agent. In some preferred embodiments, the reaction temperature in step (c) and / or (e) is in the range of 25-140 °C. In some preferred embodiments, the reaction time in step (c) and / or (e) is in the range of 2-72 hours. In some preferred embodiments, the solvent used in step (c) and / or (e) is selected from water, methanol, ethanol, acetonitrile, benzene, xylene, acetone, N,N'-dimethylformamide, DMSO, and the like.

[0124] In some preferred embodiments, the hydrolysis reaction in step (d) and / or (f) is carried out with a base selected from NaOH, LiOH, and the like. In some preferred embodiments, the reaction temperature in step (d) and / or (f) is in the range of 0-60 °C. In some preferred embodiments, the reaction time in step (d) and / or (f) is in the range of 0.5-2 hours. In some preferred embodiments, the solvent used in step (d) and / or (f) is selected from water, methanol, ethanol, tetrahydrofuran, N,N'-dimethylformamide, and the like.

[0125] In particular, the present application also provides the use of each intermediate (e.g. the compound of formula I-1, the compound of formula I-4, the compound of formula F) in the above-mentioned preparation methods for the preparation of the compound of general formula (I) as described in the present application.

[0126] Throughout this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0127] As used herein, the term "pharmaceutically acceptable salt" refers to (1) salts formed between an acidic functional group (e.g., -COOH, -OH, -SO3H, etc.) present in the compounds disclosed herein and a suitable inorganic or organic cation (base), such as salts formed between the compounds disclosed herein and an alkali metal or alkaline earth metal, ammonium salts of the compounds disclosed herein, and salts formed between the compounds disclosed herein and a nitrogen-containing organic base; and (2) salts formed between a basic functional group (e.g., -NH2, etc.) present in the compounds disclosed herein and a suitable inorganic or organic anion (acid), such as salts formed between the compounds disclosed herein and an inorganic acid or an organic carboxylic acid.

[0128] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0129] In this document, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon group. 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 Alkyl", "C 1-2 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0130] In this context, the term "alkylene" refers to a group obtained by losing two hydrogen atoms from a straight-chain or branched alkane, such as C 1-6 Alkylene, C 1-4 Alkylene, C 1-2 Specific examples include, but are not limited to, methylene, 1,2-ethylene, 1,2-ethylene, 1,3-propylene, 1,4-butylene, and the like.

[0131] As used herein, the term "alkoxy" refers to a group of the structure alkyl-O-, wherein alkyl is as defined above. 1-6 Alkoxy", "C 1-4 Alkoxy", "C1-3 alkoxy", "C 1-2 alkoxy", "C

[0132] In the present text, the term "halo-C 1-6 alkyl" means a group resulting from the replacement of at least one hydrogen atom (e.g. 1, 2 or 3) in a C 1-6 alkyl group as previously described with a halogen atom (e.g. fluorine or chlorine). Particular examples include but are not limited to fluoromethyl, difluoromethyl, trifluoromethyl and the like.

[0133] In the present text, the term "alkenyl" means a straight-chain or branched alkenyl group containing at least one double bond, including for example "C 2-6 alkenyl", "C 2-4 alkenyl" and the like. Particular examples include but are not limited to: ethenyl, 1 -propenyl, 2-propenyl, 1 -butenyl, 2-butenyl, 1,3- butadienyl, 1 -pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl and the like.

[0134] In the present text, the term "alkenylene" means a straight-chain or branched alkenylene group resulting from the loss of two hydrogen atoms from an alkenyl group, for example C 2-6 alkenylene, C 2-4 alkenylene. Particular examples include but are not limited to ethenylene, 1,3- propenylene and the like.

[0135] In the present text, the term "carbocyclic" means a cyclic structure in which all ring members are carbon atoms, including aliphatic carbocyclic rings and aromatic rings. The aliphatic carbocyclic rings are saturated or partially unsaturated cyclic structures which do not have aromatic character. Examples include 3-7 membered aliphatic carbocyclic rings, 3-6 membered aliphatic carbocyclic rings, 4-6 membered aliphatic carbocyclic rings, 5-6 membered aliphatic carbocyclic rings and the like. Particular examples include but are not limited to: cyclopropane ring, cyclobutane ring, cyclopentane ring, cyclohexane ring, cycloheptane ring, cyclooctane ring and the like.

[0136] As used herein, the term "aliphatic heterocycle" refers to a cyclic group without aromatic characteristics in which at least one ring atom is a heteroatom, wherein the heteroatom is selected from nitrogen, oxygen, and sulfur atoms. The aliphatic heterocycle includes monocyclic or polycyclic aliphatic heterocycles. The monocyclic aliphatic heterocycle can be a 3-7 membered (e.g., 3-6 membered, 5-6 membered, 3, 4, 5, 6, or 7 membered) saturated or partially unsaturated monocyclic aliphatic heterocycle containing 1-5 (e.g., 1-2) heteroatoms selected from nitrogen, oxygen, and sulfur. Specific examples include, but are not limited to, oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, and the like.

[0137] The polycyclic heterocyclic ring can be, for example, a 6-12-membered polycyclic heterocyclic ring containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur, a 6-9-membered polycyclic heterocyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur, a 6-12-membered bicyclic heterocyclic ring containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur, a 7-10-membered bicyclic heterocyclic ring containing 1-5 heteroatoms selected from nitrogen, oxygen and sulfur, or a 6-9-membered bicyclic heterocyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur. The bicyclic heterocyclic ring can be a spiro heterocyclic ring, a bridged heterocyclic ring or a fused heterocyclic ring. The spiro heterocyclic ring is formed by two rings sharing one carbon atom, such as a 6-9-membered spiro heterocyclic ring containing 1-3 (e.g., 1-2) heteroatoms of nitrogen, oxygen and sulfur. Specific examples include, but are not limited to: wait.

[0138] The bridged heterocycle is formed by two or more rings sharing two non-adjacent ring atoms, such as a 6-9 membered bridged heterocycle with 1-3 heteroatoms selected from nitrogen, oxygen and sulfur. Specific examples include but are not limited to: wait.

[0139] The heterocyclic ring is formed by two or more rings sharing two adjacent atoms, for example, a 6-9 membered heterocyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur. Specific examples include but are not limited to: wait.

[0140] As used herein, the term "aromatic heterocycle" refers to a cyclic group having aromaticity containing at least one heteroatom selected from nitrogen, oxygen, and sulfur in the ring atoms, including monocyclic aromatic heterocycles and polycyclic aromatic heterocycles (e.g., bicyclic aromatic heterocycles). For example, a 5-10 membered heteroaromatic ring containing 1-5 heteroatoms selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring containing 1-4 (e.g., 1-2) heteroatoms selected from nitrogen, oxygen, and sulfur, and an 8-10 membered bicyclic heteroaromatic ring containing 1-4 (e.g., 1-2) heteroatoms selected from nitrogen, oxygen, and sulfur. Preferably, the heteroatom is nitrogen. Specific examples include, but are not limited to, a furan ring, a thiophene ring, a pyrrole ring, a thiazole ring, an isothiazole ring, a thiadiazole ring, an oxazole ring, an isoxazole ring, an oxadiazole ring, an imidazole ring, a pyrazole ring, a 1,2,3-triazole ring, a 1,2,4-triazole ring, a 1,2,3-oxadiazole ring, a 1,2,4-oxadiazole ring, a 1,2,5-oxadiazole ring, a 1,3,4-oxadiazole ring, a pyridine ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a 1,2,3-triazine ring, a 1,3,5-triazine ring, a 1,2,4,5-tetrazine ring, a benzimidazolyl ring, an imidazopyridyl ring, a quinolyl ring, an isoquinolyl ring, a cinnolinyl ring, a quinazolinyl ring, a quinoxalinyl ring, and a naphthyridinyl ring.

[0141] As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity, such as a 6-10 membered aryl group, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 It shows that chidamide synergizes with Example 3 to inhibit the IDO enzyme activity of HeLa cells.

[0143] Figure 2 It shows that chidamide synergizes with Example 20 to inhibit the IDO enzyme activity of HeLa cells.

[0144] Figure 3 It was shown that chidamide synergistically inhibited the IDO enzyme activity of HeLa cells with Example 72.

[0145] Figure 4 It shows that chidamide synergizes with Example 87 to inhibit the IDO enzyme activity of HeLa cells.

[0146] Figure 5 The graph shows the inhibition curve of the combined use of chidamide and Example 3 on the growth of CT-26 tumor in mice.

[0147] Figure 6 The results show that the combined use of chidamide and Example 3 inhibits the weight of CT-26 tumors in mice.

[0148] Figure 7 The graph shows the inhibition curve of the combined use of chidamide and Example 20 on the growth of CT-26 tumors in mice.

[0149] Figure 8 The inhibition curve of chidamide in combination with Example 72 on the growth of CT-26 tumor in mice is shown.

[0150] Figure 9 The inhibition curve of chidamide in combination with Example 87 on the growth of CT-26 tumor in mice is shown. DETAILED DESCRIPTION

[0151] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. Hereinafter, if not specifically stated, the materials and methods of operation used in the present application are well known in the art. If not noted specifically in the examples, the operations are carried out under the conventional conditions or the conditions recommended by the manufacturer. If not noted specifically, the reagents or instruments used are all conventional products that can be obtained commercially.

[0152] The full names represented by the abbreviations of the reagents used in the examples are as follows:

[0153] KHMDS Potassium hexamethyldisilazide

[0154] HATU 2-(7-oxadiazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0155] DIPEA Diisopropylethylamine

[0156] HOBt 1-Hydroxybenzotriazole

[0157] EDCI.HCl 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0158] DMF N,N-Dimethylformamide

[0159] THF Tetrahydrofuran

[0160] EA Ethyl acetate

[0161] DCM Dichloromethane

[0162] Example 1 Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxylate

[0163]

[0164] Tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (2.04 g, 9.6 mmol) was dissolved in dioxane (16 mL) and stirred at room temperature. KHMDS (9.6 mL, 9.6 mmol) was added and stirred at room temperature for 5 minutes. Then, a solution of 4-chloro-6-fluoroquinoline (1.46 g, 8.0 mmol) in dioxane (5 mL) was added. The reaction mixture was stirred at 60°C for 30 minutes, then the temperature was removed, cooled to room temperature, and stirred for 15 hours. The mixture was then poured into water and the pH was adjusted to 8-10 with aqueous NaHCO3. The mixture was extracted with ethyl acetate, and the combined organic phases were dried, filtered, and distilled under reduced pressure to give a residue, which was then separated by column chromatography [ethyl acetate / petroleum ether = 3 / 2] to obtain light yellow viscous oily product tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxylate (1.3 g, yield: 45%), LC-MS (m / z) 359 (M+1).

[0165] Example 2 Preparation of 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride

[0166]

[0167] Tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxylate (1.3 g, 3.6 mmol) was dissolved in 20 mL of dichloromethane, and concentrated hydrochloric acid (1 mL) was added. The reaction solution was stirred at room temperature for 16 hours and then concentrated directly to obtain 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (1.0 g, yield: 94%) as an off-white solid. LC-MS (m / z) 259 (M+1).

[0168] Example 3 Preparation of N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide

[0169]

[0170] p-Chloroaniline (50 mg, 0.39 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL). Triphosgene (116 mg, 0.39 mmol) was added under ice-cooling, followed by the slow dropwise addition of triethylamine (0.2 mL, 1.6 mmol). The reaction mixture was stirred under ice-cooling for 25 minutes, then concentrated under reduced pressure, and ultra-dry N,N-dimethylformamide (5 mL) was added. A solution of 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (115 mg, 0.43 mmol) in N,N-dimethylformamide (1 mL) was then added under ice-cooling, followed by the slow dropwise addition of triethylamine (0.3 mL, 2.3 mmol). The reaction solution was stirred in an ice bath for 10 minutes, the ice bath was removed, and the reaction solution was stirred at room temperature for 40 minutes, then poured into water to precipitate a solid, which was filtered, washed with water, and dried to obtain a solid. Column chromatography [methanol / dichloromethane = 1 / 20] was used to obtain N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide (120 mg, yield: 70%) as a white solid.

[0171] 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=5.1Hz,1H),8.55(s,1H),8.02(dd,J=9.2,5.4Hz,1H),7.79(dd,J=9.7,3.0Hz,1H),7.69–7.62(m,1H),7.56– 7.50(m,2H),7.30–7.24(m,2H),6.92(d,J=5.2Hz,1H),4.97(p,J=6.6H z,1H),4.06(s,2H),4.01(s,2H),2.95–2.84(m,2H),2.47-2.39(m,2H).

[0172] LC-MS (m / z) 412 (M+1).

[0173] Example 4 Preparation of N-(3-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0174]

[0175] White solid N-(3-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-carboxamide (32 mg, 57% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (40 mg, 0.14 mmol) and 3-methoxyaniline (18 mg, 0.14 mmol) according to a similar procedure as in Example 3.

[0176] 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=5.1Hz,1H),8.41(s,1H),8.02(dd,J=9.2,5.4Hz,1H ),7.79(dd,J=9.7,2.9Hz,1H),7.66(td,J=8.8,3.0Hz,1H),7.19(t,J=2.2Hz,1H),7.1 4–7.03(m,2H),6.92(d,J=5.3Hz,1H),6.49(ddd,J=8.0,2.7,1.2Hz,1H),4.97(p,J=6. 7Hz,1H),4.05(s,2H),3.99(s,2H),3.69(s,3H),2.92–2.81(m,2H),2.49–2.36(m,2H).

[0177] LC-MS (m / z) 408 (M+1).

[0178] Example 5 Preparation of N-(4-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0179]

[0180] White solid N-(4-methoxyphenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-carboxamide (26 mg, 47% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (40 mg, 0.14 mmol) and 4-methoxyaniline (18 mg, 0.14 mmol) according to a similar procedure as in Example 3.

[0181] 1 H NMR(400MHz, DMSO-d6)δ8.70(d,J=5.1Hz,1H),8.25(s,1H),8.02(dd,J=9.4, 5.3Hz,1H),7.83–7.75(m,1H),7.66(t,J=8.8Hz,1H),7.37(d,J=8.7Hz,2H), 6.92(d,J=5.2Hz,1H),6.81(d,J=8.6Hz,2H),5.02–4.92(m,1H),4.03(s,2H) ,3.97(s,2H),3.69(s,3H),3.32(s,5H),2.87(s,2H),2.44(d,J=6.7Hz,2H).

[0182] LC-MS (m / z) 408 (M+1).

[0183] Example 6 Preparation of (4-chlorophenyl)(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)methanone

[0184]

[0185] 4-((2-Azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (60 mg, 0.2 mmol) was dissolved in 10 ml of DMF, and p-chlorobenzoic acid (38.2 mg, 0.24 mmol), EDCI (77.8 mg, 0.4 mmol), HOBt (55 mg, 0.4 mmol), and 1 ml of DIPEA were added. The reaction mixture was stirred at room temperature for 16 hours. The reaction solution was poured into 40 ml of ice water and extracted twice with ethyl acetate (20 ml). The organic phases were combined, washed with saturated brine (40 ml), dried over anhydrous sodium sulfate, and then separated by column chromatography [dichloromethane / methanol = 20 / 1] to obtain a white solid (20 mg, 25% yield).

[0186] 1 H NMR (400MHz, DMSO-d6) δ8.68(d,J=5.1Hz,1H),8.01(dd,J=9.2,5.4Hz,1H),7.76(t,J=7.4Hz,1H),7.70–7.59(m,3H),7.51(t,J= 9.2Hz,2H),6.87(dd,J=17.2,5.2Hz,1H),4.93(p,J=6.6Hz,1H),3.54–3.40(m,4H),2.88(t,J=9.9Hz,2H),2.43(d,J=8.5Hz,2H).

[0187] LC-MS (m / z) 397 (M+1).

[0188] Example 7 Preparation of (E)-3-(4-chlorophenyl)-1-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)prop-2-en-1-one

[0189]

[0190] White solid (E)-3-(4-chlorophenyl)-1-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)prop-2-en-1-one (18 mg, 21% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (40 mg, 0.14 mmol) and 4-chlorocinnamic acid (18 mg, 0.14 mmol) according to a similar procedure as in Example 6.

[0191] 1 H NMR (400MHz, DMSO-d6) δ8.71(d,J=5.1Hz,1H),8.03(dd,J=9.2,5.4Hz,1H),7.92(td,J=6.4,3.7Hz,1H),7 .80(dd,J=9.7,2.9Hz,1H),7.75(dd,J=15.6,1.7Hz,1H),7.66(td,J=8.8,3.0Hz,1H),7.57–7.49(m,1H),7 .47–7.35(m,2H),6.92(t,J=4.9Hz,1H),6.77(dd,J=15.6,8.5Hz,1H),4.99(p,J=6.6Hz,1H),4.40(d,J=2 7.1Hz, 2H), 4.07 (d, J=27.4Hz, 2H), 2.92 (ddd, J=13.1, 6.2, 2.6Hz, 2H), 2.46 (ddt, J=9.7, 6.2, 3.2Hz, 2H).

[0192] LC-MS (m / z) 423 (M+1).

[0193] Example 8 Preparation of p-Nitrocinnamyl Methyl Ether

[0194]

[0195] p-Nitrocinnamyl alcohol (200 mg, 1.1 mmol) was dissolved in 20 ml of dry tetrahydrofuran and stirred under nitrogen until completely dissolved. Sodium hydride (178 mg, 4.4 mmol) was added and stirred for 20 minutes. Methyl iodide (174 mg, 1.2 mmol) was then added and reacted at room temperature for 16 hours. The reaction solution was poured into 20 ml of ice water and extracted twice with ethyl acetate (20 ml). The mixture was dried over anhydrous sodium sulfate, and the organic phase was dried and separated by solid column chromatography [ethyl acetate / petroleum ether = 1 / 2] to obtain a light yellow solid (60 mg, 28% yield).

[0196] Example 9 Preparation of p-aminocinnamyl alcohol methyl ether

[0197]

[0198] 4-Nitrocinnamyl methyl ether (60 mg, 0.31 mmol) was dissolved in 10 ml of ethanol, iron powder (52 mg, 0.93 mmol) was added, stirred for 10 minutes, heated under reflux for 2 hours, poured into ice water (20 ml), and the pH was adjusted to 8 with sodium bicarbonate. The mixture was extracted twice with ethyl acetate (20 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain a residue, which was then separated by column chromatography [ethyl acetate / petroleum ether = 3 / 2] to obtain a brown solid (20 mg, 39% yield), LC-MS (m / z) 164 (M+1).

[0199] Example 10 Preparation of (E)-(6-((6-fluoroquinolin-4-yl)oxy)-N-(4-(3-methoxyprop-1-en-1-yl)phenyl)-2-azaspiro[3.3]heptane-2-carboxamide

[0200]

[0201] White solid (E)-(6-((6-Fluoroquinolin-4-yl)oxy)-N-(4-(3-methoxyprop-1-en-1-yl)phenyl)-2-azaspiro[3.3]hept-2-carboxamide (6 mg, 11% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (36 mg, 0.12 mmol) and p-aminocinnamyl methyl ether (20 mg, 0.12 mmol) according to a similar procedure as in Example 3.

[0202] 1H NMR (400MHz, DMSO-d6) δ8.71(d,J=5.1Hz,1H),8.48(s,1H),8.03(dd,J=9.3,5.4Hz,1H),7.80(dd ,J=9.7,3.0Hz,1H),7.66(ddd,J=9.3,8.4,3.0Hz,1H),7.48(d,J=8.7Hz,2H),7.32(d,J=8.6Hz,2H ),6.93(d,J=5.2Hz,1H),6.51(d,J=16.2Hz,1H),6.20(dt,J=16.0,6.0Hz,1H),5.76(s,2H),5.07 –4.89(m,1H),4.07(s,2H),4.04–3.94(m,2H),3.27(s,3H),2.95–2.76(m,2H),2.48–2.34(m,2H).

[0203] LC-MS (m / z) 448 (M+1).

[0204] Example 11 Preparation of 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-azacyclo[3.3]hept-2-yl)-2-(anilino)ethyl-1-one

[0205]

[0206] White solid 1-(6-((6-Fluoroquinolin-4-yl)oxy)-2-azacyclo[3.3]hept-2-yl)-2-(anilino)ethyl-1-one (35 mg, 52% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (50 mg, 0.17 mmol) and 2-(anilino)acetic acid (30.8 mg, 0.2 mmol) according to a similar procedure as in Example 6.

[0207] 1 H NMR (400MHz, DMSO-d6) δ8.69(d,J=5.1Hz,1H),8.02(dd,J=9.4,5.4Hz,1H),7.77(t,J=6.9Hz,1H),7.64(t,J=8.6Hz,1H),7.08(q,J=7.5Hz,2H),6.92–6 .86(m,1H),6.62–6.52(m,2H),5.75(s,2H),5.00–4.89(m,1H),4.02(s,2H) ,3.94(s,2H),3.67(d,J=10.4Hz,2H),2.94–2.80(m,2H),2.45–2.40(m,2H).

[0208] LC-MS (m / z) 492 (M+1).

[0209] Example 12 Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-3-azacyclo[3.1.0]hexane-3-carboxylate

[0210]

[0211] Tert-butyl 6-hydroxy-3-azabicyclo[3.1.0]hexane-3-carboxylate (603 mg, 3.03 mmol) was dissolved in 30 ml of DMF. Sodium hydride (550 mg, 13.7 mmol) was added under ice-bath and stirred for 30 minutes. 6-Fluoro-4-chloroquinoline (500 mg, 2.75 mmol) was then added and the temperature was raised to 75°C for 2 hours. The mixture was poured into ice water (20 ml) and extracted twice with ethyl acetate (20 ml). The mixture was dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography (ethyl acetate: petroleum ether = 1:1) to give a white solid (230 mg, 24% yield). LC-MS (m / z) 345 (M+1).

[0212] Example 13 Preparation of 4-((3-azacyclo[3.1.0]hex-4-yl)oxy)-6-fluoroquinoline hydrochloride

[0213]

[0214] Tert-butyl 6-((6-fluoroquinolin-4-yl)oxy)-3-azacyclo[3.1.0]hexane-3-carboxylate (50 mg, 0.145 mmol) was dissolved in 20 mL of dichloromethane, and concentrated hydrochloric acid (0.5 mL) was added. After stirring at room temperature for 3 hours, the mixture was directly concentrated to obtain a white solid (38.8 mg, 95% yield). LC-MS (m / z) 245 (M+1).

[0215] Example 14 Preparation of 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-azacyclo[3.1.0]hept-3-yl)-2-(anilino)ethyl-1-one

[0216]

[0217] White solid 1-(6-((6-fluoroquinolin-4-yl)oxy)-2-azacyclo[3.1.0]hept-3-yl)-2-(anilino)ethyl-1-one (60 mg, 89% yield) was prepared from 4-((3-azacyclo[3.1.0]hexan-4-yl)oxy)-6-fluoroquinoline hydrochloride (50 mg, 0.18 mmol) and 2-(anilino)acetic acid (33.7 mg, 0.21 mmol) according to a similar procedure as in Example 6.

[0218] 1 H NMR (400MHz, DMSO-d6) δ8.47(d,J=5.2Hz,1H),8.01(dd,J=11.0,2.8Hz,1H),7.86(dd,J=9.2,5 .8Hz,1H),7.52(ddd,J=9.3,8.1,2.7Hz,1H),7.43(s,1H),7.09(dd,J=8.4,7.1Hz,2H),6.74(d ,J=5.2Hz,1H),6.68–6.61(m,2H),6.56(tt,J=7.2,1.1Hz,1H),5.53(t,J=5.1Hz,1H),3.99(d, J=10.4Hz,1H),3.91–3.86(m,2H),3.78–3.69(m,2H),3.53–3.47(m,2H),2.32(d,J=2.4Hz,2H).

[0219] LC-MS (m / z) 377 (M+1).

[0220] Example 15 Preparation of N-(4-chlorobenzyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0221]

[0222] White solid N-(4-chlorobenzyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-carboxamide (20 mg, 14% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (100 mg, 0.36 mmol) and 4-chlorobenzylamine (40 mg, 0.36 mmol) according to a similar procedure as in Example 3.

[0223] 1 H NMR (400MHz, DMSO-d6) δ8.72(d,J=5.1Hz,1H),8.04(dd,J=9.2,5.4Hz,1H),7. 80(dd,J=9.7,3.0Hz,1H),7.67(td,J=8.8,3.0Hz,1H),7.42–7.35(m,2H),7.29 (d,J=8.4Hz,2H),6.94(dd,J=7.7,5.5Hz,2H),4.97(p,J=6.7Hz,1H),4.20(d, J=6.1Hz,2H),3.96(s,2H),3.89(s,2H),2.91–2.80(m,2H),2.46–2.34(m,2H).

[0224] LC-MS (m / z) 426 (M+1).

[0225] Example 16 Preparation of N-(4-fluorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0226]

[0227] White solid N-(4-fluorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-carboxamide (20 mg, 14% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (100 mg, 0.36 mmol) and 4-fluoroaniline (40 mg, 0.36 mmol) according to a similar procedure as in Example 3.

[0228] 1H NMR (400MHz, DMSO-d6) δ8.70(d,J=5.1Hz,1H),8.47(s,1H),8.03(dd,J=9.2,5.4Hz,1H),7.79(dd,J=9.7,3.0Hz,1H),7.66(m,1H),7. 49(m,2H),7.05(m,2H),6.92(d,J=5.1Hz,1H),4.97(t,J=6.7Hz,1H),4.05(s,2H),4.00(s,2H),2.91–2.86(m,2H),2.46–2.41(m,2H).

[0229] LC-MS (m / z) 396 (M+1).

[0230] Example 17 Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.4]octane-2-carboxylate

[0231]

[0232] 4-Chloro-6-fluoroquinoline (181 mg, 1.0 mmol), tert-butyl-6-amino-2-azoazole [3.4] octanoic acid-2-carboxylate (226 mg, 1.0 mmol) and cesium carbonate (980 mg, 3.0 mmol) were dissolved in dioxane (5 mL), and then 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (48 mg, 0.1 mmol) and tris (dibenzylideneacetone) dipalladium (46 mg, 0.05 mmol) were added under nitrogen protection. The reaction solution was heated at 123° C. and stirred for 7 hours, then poured into water, extracted with ethyl acetate, washed with water, and the organic phase was concentrated under reduced pressure to obtain a residue. Column chromatography was used to obtain tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.4]octan-2-carboxylate (100 mg, 27% yield) as a light yellow oil. LC-MS (m / z) 372 (M+1)

[0233] Example 18 Preparation of 4-(2-azaspiro[3.4]oct-6-yl)amino-6-fluoroquinoline hydrochloride

[0234]

[0235] Tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.4]octan-2-carboxylate (100 mg, 0.27 mmol) was dissolved in 5 mL of dichloromethane, and concentrated hydrochloric acid (1 mL) was added. The reaction mixture was stirred at room temperature for 16 hours and then concentrated to afford 4-(2-azaspiro[3.4]octan-6-yl)amino-6-fluoroquinoline hydrochloride (80 mg, 94% yield) as an off-white solid. LC-MS (m / z) 272 (M+1).

[0236] Example 19 Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.4]octan-2-carboxamide

[0237]

[0238] p-Chloroaniline (30 mg, 0.24 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL). Triphosgene (70 mg, 0.24 mmol) was added under ice-cooling, followed by the slow dropwise addition of triethylamine (0.2 mL, 1.6 mmol). The reaction mixture was stirred under ice-cooling for 25 minutes, then concentrated under reduced pressure, and ultra-dry N,N-dimethylformamide (5 mL) was added. A solution of 4-(2-azaspiro[3.4]octan-6-yl)amino-6-fluoroquinoline hydrochloride (65 mg, 0.21 mmol) in N,N-dimethylformamide (1 mL) was then added under ice-cooling, followed by the slow dropwise addition of triethylamine (0.3 mL, 2.3 mmol). The reaction solution was stirred in an ice bath for 10 minutes, the ice bath was removed, and the reaction solution was stirred at room temperature for 40 minutes. The solution was then poured into water to precipitate a solid, which was filtered, washed with water, and dried to obtain a solid. The solid was then separated by column chromatography [methanol / dichloromethane = 1 / 20] to obtain N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.4]octan-2-carboxamide (68 mg, 70% yield) as a white solid.

[0239] 1 H NMR (400MHz, DMSO-d6) δ8.53 (s, 1H), 8.41 (d, J = 5.3Hz, 1H), 8.18 (dd, J = 11.2, 2. 9Hz,1H),7.85(dd,J=9.2,5.8Hz,1H),7.54(d,J=9.0Hz,2H),7.28(d,J=8.9Hz,2H ),6.81(d,J=6.0Hz,1H),6.51(d,J=5.4Hz,1H),3.98–3.81(m,3H),2.43–2.33(m, 1H),2.21–2.11(m,2H),2.10–1.96(m,2H),1.96–1.84(m,2H),1.83–1.73(m,2H).

[0240] LC-MS (m / z) 425 (M+1).

[0241] Example 20 Preparation of N-(Benzo[d][1,3]dioxolan-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0242]

[0243] White solid N-(benzo[d][1,3]dioxolan-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-carboxamide (45 mg, 45% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (65 mg, 0.22 mmol) and 3,4-methylenedioxyaniline (33 mg, 0.242 mmol) according to a similar procedure as in Example 3.

[0244] 1 H NMR(400MHz,DMSO-d6)δ8.70(d,J=5.1Hz,1H),8.31(s,1H),8.02(dd,J=9.2,5.4Hz,1 H),7.79(dd,J=9.7,3.0Hz,1H),7.66(td,J=8.8,3.0Hz,1H),7.18(d,J=2.0Hz,1H),6. 92(d,J=5.2Hz,1H),6.85(dd,J=8.4,2.1Hz,1H),6.77(d,J=8.4Hz,1H),5.93(s,2H),4 .97(p,J=6.6Hz,1H),4.03(s,2H),3.97(s,2H),2.94–2.79(m,2H),2.47–2.38(m,2H).

[0245] LC-MS (m / z) 422 (M+1).

[0246] Example 21 Preparation of tert-butyl 6-((quinolin-4-yl)oxy)-2-azaspiro[3.4]heptane-2-carboxylate

[0247]

[0248] Tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (0.717 g, 3.3 mmol) was dissolved in dioxane (20 mL) and stirred at room temperature. KHMDS (3.6 mL, 3.6 mmol) was added and stirred at room temperature for 30 minutes. Then, a solution of 4-chloroquinoline (0.5 g, 3.0 mmol) in dioxane (5 mL) was added. The reaction mixture was stirred at 75°C for 60 minutes, then the temperature was removed, cooled to room temperature, and stirred for 15 hours. The mixture was then poured into 20 mL of water. Extraction was performed twice with ethyl acetate (20 mL). The combined organic phases were dried, filtered, and evaporated under reduced pressure to obtain a residue. The residue was then separated by column chromatography (ethyl acetate / petroleum ether = 3 / 2) to obtain a light yellow oil (0.35 g, 33% yield). LC-MS (m / z) 341.16 (M+1) was obtained.

[0249] Example 22 Preparation of 4-(2-azaspiro[3.3]hept-6-yl)oxyquinoline hydrochloride

[0250]

[0251] Tert-butyl 6-((quinolin-4-yl)oxy)-2-azaspiro[3.4]heptane-2-carboxylate (0.35 g, 1.028 mmol) was dissolved in 20 mL of dichloromethane, and concentrated hydrochloric acid (0.5 mL) was added. After stirring at room temperature for 3 hours, the mixture was directly concentrated to obtain an off-white solid (0.235 g, 95% yield). LC-MS (m / z) 241.2 (M+1).

[0252] Example 23 Preparation of N-(4-chlorophenyl)-6-(quinolin-4-yloxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0253]

[0254] p-Chloroaniline (25.3 mg, 0.198 mmol) was dissolved in ultra-dry dichloromethane (10 mL) and, under nitrogen protection, triphosgene (21.4 mg, 0.072 mmol) was added under ice-cooling, followed by triethylamine (0.5 mL, 3.6 mmol). The mixture was stirred under ice-cooling for 25 minutes. A solution of 4-(2-azaspiro[3.3]hept-6-yl)oxyquinoline hydrochloride (50 mg, 0.18 mmol) in dichloromethane (5 mL) and triethylamine (0.5 mL) was then added under ice-cooling. The reaction mixture was stirred under ice-cooling for 10 minutes. The ice-cooling was then removed and the reaction mixture was stirred at room temperature for 120 minutes. 5 mL of methanol was added and stirred for 5 minutes. The mixture was then spin-dried and separated by solid column chromatography [methanol / dichloromethane = 1 / 20] to obtain a white solid (38 mg, 53.6% yield).

[0255] 1H NMR (400MHz, DMSO-d6) δ8.71(d,J=5.1Hz,1H),8.57(s,1H),8.17–8.10(m,1H),7.95(d,J=8.4Hz,1H),7.74(ddd,J=8.6,6.8,1.5Hz,1H),7.62– 7.48(m,3H),7.30–7.24(m,2H),6.88(d,J=5.2Hz,1H),4.97(t,J=6.6H z,1H),4.07(s,2H),4.01(s,2H),2.96–2.84(m,2H),2.48–2.37(m,2H).

[0256] LC-MS (m / z) 394 (M+1).

[0257] Example 24 Preparation of tert-butyl 6-((quinolin-3-yl)oxy)-2-azaspiro[3.4]heptane-2-carboxylate

[0258]

[0259] Tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (98.7 mg, 0.458 mmol) was dissolved in 10 ml of DMF, sodium hydride (61.1 mg, 1.5 mmol) was added and stirred for 30 minutes, 3-chloroquinoline (50 mg, 0.3 mmol) was added, the temperature was raised to 75 degrees Celsius for 1 hour, ice water (20 ml) was poured into the mixture, and the mixture was extracted twice with ethyl acetate (20 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain a residue, which was then separated by column chromatography [ethyl acetate / petroleum ether = 3 / 2] to obtain a white solid (30 mg, 49% yield), LC-MS (m / z) 341 (M+1).

[0260] Example 25 Preparation of 3-(2-azaspiro[3.3]hept-6-yl)oxyquinoline hydrochloride

[0261]

[0262] Tert-butyl 6-((quinolin-3-yl)oxy)-2-azaspiro[3.4]heptane-2-carboxylate (30 mg, 0.088 mmol) was dissolved in 20 mL of dichloromethane, and concentrated hydrochloric acid (0.5 mL) was added. After stirring at room temperature for 3 hours, the mixture was directly concentrated to obtain a white solid (23.1 mg, 95% yield). LC-MS (m / z) 241 (M+1).

[0263] Example 26 Preparation of N-(4-chlorophenyl)-6-(quinolin-3-yloxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0264]

[0265] p-Chloroaniline (13.5 mg, 0.1 mmol) was dissolved in ultra-dry dichloromethane (10 mL) and, under nitrogen protection, triphosgene (13 mg, 0.043 mmol) was added under ice-cooling, followed by triethylamine (0.5 mL, 3.6 mmol). The mixture was stirred under ice-cooling for 25 minutes. A solution of 3-(2-azaspiro[3.3]hept-6-yl)oxyquinoline hydrochloride (23.1 mg, 0.08 mmol) in dichloromethane (5 mL) and triethylamine (0.5 mL) was added under ice-cooling. The reaction mixture was stirred under ice-cooling for 10 minutes. The ice-cooling was then removed and the reaction mixture was stirred at room temperature for 120 minutes. 5 mL of methanol was added and stirred for 5 minutes. The mixture was then spin-dried and separated by solid column chromatography [methanol / dichloromethane = 1 / 20] to obtain a white solid (25 mg, 79% yield).

[0266] 1 H NMR(400MHz, DMSO-d6)δ8.60(d,J=2.8Hz,2H),7.95–7.88(m,2H),7.63–7.52(m,5H),7.27(d,J= 8.9Hz,2H),4.86–4.82(m,1H),4.06(s,2H),3.99(s,2H),2.89–2.84(m,2H),2.37–2.32(m,2H).

[0267] LC-MS (m / z) 394.2 (M+1).

[0268] Example 27 Preparation of tert-butyl 6-(quinazolin-4-yloxy)-2-azaspiro[3.3]heptane-2-carboxylate

[0269]

[0270] Dissolve tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (155.5 mg, 0.729 mmol) in 20 ml of DMF. Add sodium hydride (120 mg, 3 mmol) under ice-cooling and stir for 30 minutes. Then add 3-chloroquinoline (100 mg, 0.6 mmol). React at room temperature for 0.5 hour. Pour into ice water (30 ml) and extract twice with ethyl acetate (20 ml). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and evaporate to dryness under reduced pressure to obtain a brown liquid (120 mg, 58% yield). LC-MS (m / z) 342 (M+1).

[0271] Example 28 Preparation of 4-(2-azaspiro[3.3]hept-6-yl)oxyquinazoline trifluoroacetate

[0272]

[0273] Tert-butyl 6-(quinazolin-4-yloxy)-2-azaspiro[3.3]heptane-2-carboxylate (70 mg, 0.2 mmol) was dissolved in 20 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). After stirring at room temperature for 3 hours, the mixture was concentrated to afford a gray liquid (58.7 mg, 95% yield). LC-MS (m / z) 241 (M+1).

[0274] Example 29 Preparation of N-(4-chlorophenyl)-6-(quinazolin-4-yloxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0275]

[0276] p-Chloroaniline (31.3 mg, 0.24 mmol) was dissolved in ultra-dry dichloromethane (10 mL) and, under nitrogen protection, triphosgene (24 mg, 0.08 mmol) was added under ice-cooling, followed by triethylamine (0.5 mL, 3.6 mmol). The mixture was stirred under ice-cooling for 25 minutes. A solution of 4-(2-azaspiro[3.3]hept-6-yl)oxyquinazoline trifluoroacetate (58.7 mg, 0.195 mmol) in dichloromethane (5 mL) and triethylamine (0.5 mL) was then added under ice-cooling. The reaction mixture was stirred under ice-cooling for 10 minutes. The ice-cooling was then removed and the reaction mixture was stirred at room temperature for 120 minutes. 5 mL of methanol was added and stirred for 5 minutes. The mixture was then spin-dried and separated by solid column chromatography [methanol / dichloromethane = 1 / 20] to obtain a white solid (30 mg, 39% yield).

[0277] 1 H NMR (400MHz, DMSO-d6) δ8.78(s,1H),8.56(s,1H),8.21–8.13(m,1H),8.01–7.86(m,2H),7.70(ddd,J=8.2,6.6,1.5Hz,1H) ,7.57–7.49(m,2H),7.31–7.20(m,2H),5.40(p,J=6.9Hz,1H),5.00(d,J=6.2Hz,4H),2.90–2.80(m,2H),2.49–2.42(m,2H).

[0278] LC-MS (m / z) 395 (M+1).

[0279] Example 30 Preparation of N-(4-fluorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-3-azacyclo[3.1.0]hexane-3-carboxamide

[0280]

[0281] p-Fluoroaniline (19.3 mg, 0.174 mmol) was dissolved in ultra-dry dichloromethane (10 mL). Triphosgene (17.2 mg, 0.058 mmol) was added under nitrogen atmosphere under ice-cooling, followed by triethylamine (0.5 mL, 3.6 mmol). The mixture was stirred under ice-cooling for 25 minutes. A solution of 4-((3-azacyclo[3.1.0]hex-4-yl)oxy)-6-fluoroquinoline hydrochloride (38.8 mg, 0.138 mmol) in dichloromethane (5 mL) and triethylamine (0.5 mL) was added under ice-cooling. The reaction mixture was stirred under ice-cooling for 10 minutes. The ice-cooling was then removed and the reaction mixture was stirred at room temperature for 120 minutes. 5 mL of methanol was added and stirred for 5 minutes. The mixture was then spin-dried and separated by solid column chromatography [methanol / dichloromethane = 1 / 20] to obtain a white solid (18 mg, 34% yield).

[0282] 1 H NMR (400MHz, DMSO-d6) δ8.77(d,J=5.1Hz,1H),8.23(s,1H),8.04(dd,J=9.2,5.4Hz,1H),7.73(dd,J=9.7,2.9Hz,1H),7.70–7.62(m,1H),7.56–7.47(m, 2H),7.30(d,J=5.1Hz,1H),7.11–7.02(m,2H),3.97(d,J=1.5Hz,1H),3.92( d,J=10.5Hz,2H),3.56(dt,J=10.3,2.3Hz,2H),2.17(dt,J=3.0,1.5Hz,2H).

[0283] LC-MS (m / z) 382 (M+1).

[0284] Example 31 Preparation of N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-3-azacyclo[3.1.0]hexane-3-carboxamide

[0285]

[0286] To a solution of 4-((3-azabicyclo[3.1.0]hexan-3-yl)oxy)-6-fluoroquinoline hydrochloride (45 mg, 0.15 mmol) and 2,3-dihydrobenzofuran-5-amine (24.9 mg, 0.18 mmol) in dichloromethane (5 mL) and triethylamine (0.5 mL) was added 4-chloroaniline (22.3 mg, 0.174 mmol) under nitrogen protection. The reaction mixture was stirred at room temperature for 120 min. The reaction mixture was concentrated and purified by column chromatography [methanol / dichloromethane = 1 / 20] to give N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2- carboxamide (25 mg, 44.9% yield) as a white solid.

[0287] 1 H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 5.1 Hz, 1H), 8.32 (s, 1H), 8.04 (dd, J = 9.2, 5.4 Hz, 1H), 7.72 (dd, J = 9.7, 2.9 Hz, 1H), 7.70 - 7.62 (m, 1H), 7.61 - 7.54 (m, 2H), 7.32 - 7.25 (m, 3H), 3.97 (t, J = 1.5 Hz, 1H), 3.93 (d, J = 10.5 Hz, 2H), 3.57 (dt, J = 10.5, 2.2 Hz, 2H), 2.16 (dt, J = 3.0, 1.5 Hz, 2H).

[0288] LC-MS (m / z) 398 (M+l).

[0289] Example 32 Preparation of N-(2,3-dihydrobenzofuran-5-yl)-6-((6-fluoroquinolin-4- yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0290]

[0291] Preparation of N-(2,3-dihydrobenzofuran-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2- azaspiro[3.3]heptane-2-carboxamide (55 mg, 85% yield) was prepared from 4-((2- azaspiro[3.3]heptan-6-yl)oxy)-6-fluoroquinoline hydrochloride (45 mg, 0.15 mmol) and 2,3-dihydrobenzofuran-5-amine (24.9 mg, 0.18 mmol) following a similar procedure as in Example 3.

[0292] 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=5.1Hz,1H),8.18(s,1H),8.02(dd,J=9.2,5.4Hz,1H),7. 79(dd,J=9.7,3.0Hz,1H),7.69–7.60(m,1H),7.35(d,J=2.2Hz,1H),7.09(dd,J=8.6,2.3Hz, 1H),6.92(d,J=5.2Hz,1H),6.62(d,J=8.5Hz,1H),4.97(p,J=6.6Hz,1H),4.46(t,J=8.7Hz, 2H),4.02(s,2H),3.96(s,2H),3.12(t,J=8.6Hz,2H),2.92–2.83(m,2H),2.47–2.37(m,2H).

[0293] LC-MS (m / z) 420 (M+1).

[0294] Example 33 Preparation of N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0295]

[0296] White solid N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-carboxamide (35 mg, 52% yield) was prepared from 4-((2-azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (45 mg, 0.15 mmol) and 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (27.8 mg, 0.18 mmol) according to a similar procedure as in Example 3.

[0297] 1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 5.1 Hz, 1H), 8.21 (s, 1H), 8.02 (dd, J = 9.2, 5.4 Hz, 1H), 7.79 (dd, J = 9.7, 2.9 Hz, 1H), 7.65 (td, J = 8.8, 2.9 Hz, 1H), 7.09 (d, J = 2.5 Hz, 1H), 6.97 - 6.85 (m, 2H), 6.70 (d, J = 8.7 Hz, 1H), 4.97 (p, J = 6.6 Hz, 1H), 4.25 - 4.13 (m, 4H), 4.02 (s, 2H), 3.96 (s, 2H), 2.96 - 2.80 (m, 2H), 2.48 - 2.37 (m, 2H).

[0298] LC-MS (m / z) 436 (M+l).

[0299] Example 34 Preparation of tert-butyl 6-(isoquinolin-1-yloxy)-2-azaspiro[3.3]heptane-2- carboxylate

[0300]

[0301] Tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (106 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (10 mL), sodium hydride (92 mg, 60% in mineral, 2.5 mmol) was added, the reaction was stirred at room temperature for 5 minutes, then 1-chloroisoquinoline (82 mg, 0.5 mmol) was added. The reaction was stirred at room temperature for 15 hours, quenched with water, the solid was precipitated with water, filtered, washed with water to give light yellow solid tert-butyl 6-(isoquinolin-1-yloxy)-2-azaspiro[3.3]heptane-2-carboxylate (80 mg, 47% yield), LC-MS (m / z) 341 (M+l).

[0302] Example 35 Preparation of 1-(2-azaspiro[3.3]heptan-6-yl)oxyisoquinoline hydrochloride

[0303]

[0304] Tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (106 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (10 mL), sodium hydride (92 mg, 60% in mineral, 2.5 mmol) was added, the reaction was stirred at room temperature for 5 minutes, then 1-chloroisoquinoline (82 mg, 0.5 mmol) was added. The reaction was stirred at room temperature for 15 hours, quenched with water, the solid was precipitated with water, filtered, washed with water to give light yellow solid tert-butyl 6-(isoquinolin-1-yloxy)-2-azaspiro[3.3]heptane-2-carboxylate (80 mg, 47% yield), LC-MS (m / z) 341 (M+l).

[0305] Example 36 Preparation of N-(4-chlorophenyl)-6-(isoquinolin-1-yloxy)-2-azaspiro[3.3]heptane-2-carboxamide

[0306]

[0307] p-Chloroaniline (30 mg, 0.24 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL). Triphosgene (70 mg, 0.24 mmol) was added under ice-cooling, followed by the slow dropwise addition of triethylamine (0.2 mL, 1.6 mmol). The reaction mixture was stirred under ice-cooling for 25 minutes, then concentrated under reduced pressure, and ultra-dry N,N-dimethylformamide (5 mL) was added. A solution of 1-(2-azaspiro[3.3]hept-6-yl)oxyisoquinoline hydrochloride (60 mg, 0.21 mmol) in N,N-dimethylformamide (1 mL) was then added under ice-cooling, followed by the slow dropwise addition of triethylamine (0.3 mL, 2.3 mmol). The reaction solution was stirred in an ice bath for 10 minutes, the ice bath was removed, and the reaction solution was stirred at room temperature for 40 minutes, then poured into water to precipitate a solid, which was filtered, washed with water, and dried to obtain a solid. Column chromatography [methanol / dichloromethane = 1 / 25] was used to obtain N-(4-chlorophenyl)-6-(isoquinolin-1-yloxy)-2-azaspiro[3.3]heptane-2-carboxamide (68 mg, 70% yield) as a white solid.

[0308] 1 H NMR(400MHz, DMSO-d6)δ8.56(s,1H),8.18(d,J=8.3Hz,1H),7.98(d,J=5.8Hz,1H),7.89(d,J=8.2Hz,1H),7.80–7.72(m,1H),7.63(t,J=7.6Hz,1H), 7.56–7.51(m,2H),7.38(d,J=5.9Hz,1H),7.30–7.24(m,2H),5.32(p,J=6. 9Hz,1H),4.07(s,2H),4.00(s,2H),2.88–2.76(m,2H),2.45–2.36(m,2H).

[0309] LC-MS (m / z) 394 (M+1).

[0310] Example 37 Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoate

[0311]

[0312] 4-((2-Azaspiro[3.3]hept-6-yl)oxy)-6-fluoroquinoline hydrochloride (600 mg, 2.0 mmol), ethyl 2-bromopropionate (841 mg, 4.0 mmol), and potassium carbonate (962 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL). The reaction solution was stirred at room temperature for 16 hours, then poured into water and extracted with ethyl acetate. The combined organic phases were dried, filtered, and evaporated under reduced pressure to obtain a residue, which was then separated by column chromatography [methanol / dichloromethane = 1 / 25] to obtain ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoate (450 mg, 61% yield) as a colorless oil. LC-MS (m / z) 259 (M+1).

[0313] Example 38 Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoic acid

[0314]

[0315] Ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoate (380 mg, 1.0 mmol) was dissolved in ethanol (8 mL) and water (0.5 mL), followed by the addition of sodium hydroxide (85 mg, 2.0 mmol). The reaction mixture was stirred at room temperature for 5 hours, then adjusted to pH 3-4 with concentrated hydrochloric acid. The mixture was then concentrated to afford crude 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoic acid (460 mg, crude) as an off-white solid, which was used directly in the next step. LC-MS (m / z) 331 (M+1).

[0316] Example 39 Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-yl)propanamide

[0317]

[0318] 2-(6-((6-Fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoic acid (350 mg, 1.1 mmol), p-chloroaniline (270 mg, 2.2 mmol) and N,N-diisopropylethylamine (0.66 mL, 3.6 mmol) were dissolved in N,N-dimethylformamide (15 mL), followed by the addition of 1-hydroxybenzotriazole (223 mg, 1.6 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (420 mg, 2.2 mmol). The reaction solution was stirred at room temperature for 15 hours, then poured into water, and the pH was adjusted to 8-10 with aqueous sodium bicarbonate solution. The precipitated solid was filtered, washed with water, and dried to obtain a yellow solid. Column chromatography [methanol / dichloromethane = 1 / 25] was used to obtain N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-yl)propanamide (130 mg, 28% yield) as a white solid, LC-MS (m / z) 440 (M+1).

[0319] 1 H NMR (400MHz, DMSO-d6) δ9.72 (s, 1H), 8.68 (d, J = 5.2 Hz, 1H), 8.01 (dd, J = 9.6, 5. 2Hz,1H),7.77(dd,J=9.6,2.8Hz,1H),7.72–7.62(m,3H),7.38–7.34(m,2H),6.9 0(d,J=5.2Hz,1H),4.96–4.90(m,1H),3.37–3.25(m,4H),2.97(dd,J=13.2,6.8 Hz,1H),2.84–2.77(m,2H),2.34(dd,J=13.2,6.8Hz,2H),1.11(d,J=6.8Hz,3H).

[0320] Example 40 Preparation of N-cyclohexyl-2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-yl)propanamide

[0321]

[0322] White solid N-cyclohexyl-2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanamide (56 mg, 37% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoic acid (120 mg, 0.36 mmol) and cyclohexylamine (30 mg, 0.36 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 412 (M+1).

[0323] 1 H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 5.2 Hz, 1H), 8.03 (dd, J = 9.6, 5.2 Hz, 1H), 7.78 (dd, J = 9.6, 2.8 Hz, 1H), 7.69 - 7.64 (m, 1H), 7.28 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 5.2 Hz, 1H), 4.98 - 4.92 (m, 1H), 3.56 - 3.53 (m, 1H), 3.32 - 3.23 (m, 2H), 3.21 - 3.19 (m, 2H), 2.84 - 2.70 (m, 3H), 2.33 (dd, J = 12.0, 6.8 Hz, 2H), 1.71 - 1.67 (m, 4H), 1.57 (d, J = 12.0 Hz, 1H), 1.30 - 1.12 (m, 6H), 0.99 (d, J = 6.8 Hz, 3H).

[0324] Example 41 Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptan-2-yl)-N-(pyridin-3-yl)propanamide

[0325]

[0326] White solid N-cyclohexyl-2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptan-2-yl)propanamide (15 mg, 10% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptan-2-yl)propanoic acid (120 mg, 0.36 mmol) and 3-aminopyridine (30 mg, 0.31 mmol) following a similar procedure as in Example 39, LC-MS (m / z) 407 (M+l).

[0327] 1H NMR (400MHz, DMSO-d6) δ9.81 (s, 1H), 8.82 (d, J = 2.4Hz, 1H), 8.68 (d, J = 5.2Hz, 1H), 8.26 (dd, J = 4.8 ,1.6Hz,1H),8.10–8.07(m,1H),8.01(dd,J=9.6,5.4Hz,1H),7.77(dd,J=9.6,2.8Hz,1H),7.67–7. 62(m,1H),7.33(dd,J=8.4,4.8Hz,1H),6.90(d,J=5.2Hz,1H),4.97–4.905(m,1H),3.380–3.27(m, 4H), 3.00 (q, J=6.8Hz, 1H), 2.87–2.76 (m, 2H), 2.34 (dd, J=12.0, 6.8Hz, 2H), 1.13 (d, J=6.8Hz, 3H).

[0328] Example 42 Preparation of tert-butyl 6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate

[0329]

[0330] White solid tert-butyl 6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (50 mg, 9% yield) was prepared from tert-butyl 6-(hydroxymethyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (388 mg, 1.82 mmol) and 4-chloro-6-fluoroquinoline (270 mg, 1.5 mmol) according to a procedure similar to that in Example 1. LC-MS (m / z) 359 (M+1).

[0331] Example 43 Preparation of 4-((3-azabicyclo[3.1.0]hexan-6-yl)methoxy)-6-fluoroquinoline hydrochloride

[0332]

[0333] White solid 4-((3-azabicyclo[3.1.0]hexan-6-yl)methoxy)-6-fluoroquinoline hydrochloride (50 mg, 90% yield) was prepared from tert-butyl 6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (50 mg, 0.14 mmol) according to a procedure similar to that in Example 2. LC-MS (m / z) 259 (M+1).

[0334] Example 44 Preparation of ethyl 2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate

[0335]

[0336] Ethyl 2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate (30 mg, 60% yield) as a white solid was prepared from 4-((3-azabicyclo[3.1.0]hexan-6-yl)methoxy)-6-fluoroquinoline hydrochloride (50 mg, 0.19 mmol) and ethyl 2-bromopropanoate (63 mg, 0.38 mmol) according to a procedure similar to that in Example 37. LC-MS (m / z) 359 (M+1).

[0337] Example 45 Preparation of 2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexane-3-yl)propanoic acid

[0338]

[0339] White solid 2-(6-(((6-Fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (25 mg, 92% yield) was prepared from ethyl 2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate (30 mg, 0.08 mmol) according to a procedure similar to that in Example 38. LC-MS (m / z) 331 (M+1).

[0340] Example 46 Preparation of N-(4-chlorophenyl)-2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propionamide (12659)

[0341]

[0342] Pale yellow solid N-(4-chlorophenyl)-2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propanamide (8 mg, 24% yield) was prepared from 2-(6-(((6-fluoroquinolin-4-yl)oxy)methyl)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (25 mg, 0.08 mmol) and 4-chloropropionamide (20 mg, 0.16 mmol) according to the similar procedures as in Example 39, LC-MS (m / z) 440 (M+1).

[0343] 1 H NMR(400MHz, DMSO-d6)δ9.76(s,1H),8.70(d,J=5.2Hz,1H),8.02(dd,J=9.2,5.2Hz,1H),7.78(dd ,J=9.2,3.0Hz,1H),7.70–7.61(m,3H),7.37–7.31(m,2H),7.04(d,J=5.2Hz,1H),4.25–4.07(m,2 H),3.19(q,J=6.8Hz,1H),3.01(dd,J=14.4,8.4Hz,2H),2.65(td,J=9.6,8.4,2.8Hz,1H),2.58–2 .53(m,1H),1.85(dt,J=7.2,4.4Hz,1H),1.59(tq,J=7.2,4.4,3.6Hz,2H),1.22(d,J=6.8Hz,3H).

[0344] Example 47 Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate

[0345]

[0346] Ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate (90 mg, 90% yield) as a white solid was prepared from 4-((3-azabicyclo[3.1.0]hexan-6-yl)oxy)-6-quinoline hydrochloride (80 mg, 0.29 mmol) and ethyl 2-bromopropionate (103 mg, 0.58 mmol) according to a procedure similar to that in Example 37. LC-MS (m / z) 345 (M+1).

[0347] Example 48 Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexane-3-yl)propanoic acid

[0348]

[0349] White solid 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (100 mg, 73% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate (90 mg, 0.26 mmol) according to a procedure similar to that in Example 38. LC-MS (m / z) 317 (M+1).

[0350] Example 49 Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propionamide

[0351]

[0352] White solid 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (45 mg, 36% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (100 mg, 0.32 mmol) and 4-chloroaniline (37 mg, 0.29 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 426 (M+1).

[0353] 1 H NMR(400MHz, DMSO-d6)δ9.77(s,1H),8.78(d,J=5.2Hz,1H),8.03(dd,J=9.2,5.2Hz,1H),7.74–7.61(m,4H),7.41–7.31 (m,2H),7.25(d,J=5.2Hz,1H),4.39(s,1H),3.24–3.21(m,3H),2.78–2.68(m,2H),1.93(s,1H),1.26(d,J=6.8Hz,3H).

[0354] Example 50 Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptane-2-carboxylate

[0355]

[0356] 4-Chloro-6-fluoroquinoline (271 mg, 1.5 mmol), tert-butyl 6-amino-2-azaspiro[3.3]heptane-2-carboxylate (318 mg, 1.5 mmol) and cesium carbonate (1.5 g, 4.5 mmol) were dissolved in dioxane (5 mL), and then 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (71 mg, 0.15 mmol) and tris(dibenzylideneacetone)dipalladium (70 mg, 0.075 mmol) were added under nitrogen protection. The reaction solution was heated at 123°C with stirring for 7 hours, then poured into water, extracted with ethyl acetate, washed with water, and the organic phase was concentrated under reduced pressure to obtain a residue. Column chromatography [EA] was used to obtain tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptane-2-carboxylate (280 mg, 52% yield) as a white solid. LC-MS (m / z) 358 (M+1)

[0357] Example 51 Preparation of 6-fluoro-N-(2-azaspiro[3.3]heptane-6-yl)quinolin-4-amine hydrochloride

[0358]

[0359] White solid 6-fluoro-N-(2-azaspiro[3.3]heptane-6-yl)quinolin-4-amine hydrochloride (128 mg, 94% yield) was prepared from tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptane-2-carboxylate (180 mg, 0.51 mmol) following a procedure similar to that in Example 2. LC-MS (m / z) 258 (M+1). LC-MS (m / z) 258 (M+1).

[0360] Example 52 Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)amine)-2-azaspiro[3.3]heptane-2-yl)propanoate

[0361]

[0362] Ethyl 2-(6-((6-fluoroquinolin-4-yl)amine)-2-azaspiro[3.3]heptan-2-yl)propanoate (150 mg, 83% yield) as a white solid was prepared from 6-fluoro-N-(2-azaspiro[3.3]heptan-6-yl)quinolin-4-amine hydrochloride (128 mg, 0.5 mmol) and ethyl 2-bromopropionate (100 mg, 0.55 mmol) according to a procedure similar to that in Example 37. LC-MS (m / z) 358 (M+1).

[0363] Example 53 Preparation of 2-(6-((6-fluoroquinolin-4-yl)oxy)-3-azabicyclo[3.1.0]hexane-3-yl)propanoic acid

[0364]

[0365] White solid 2-(6-((6-fluoroquinolin-4-yl)amine)-2-azaspiro[3.3]heptan-2-yl)propanoic acid (100 mg, 72% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)amine)-2-azaspiro[3.3]heptan-2-yl)propanoate (150 mg, 0.42 mmol) according to a procedure similar to that in Example 38. LC-MS (m / z) 330 (M+1).

[0366] Example 54 Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptane-2-yl)propanamide

[0367]

[0368] White solid N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptan-2-yl)propanamide (5 mg, 4% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptan-2-yl)propanoic acid (100 mg, 0.3 mmol) and 4-chloroaniline according to a procedure similar to that in Example 39. LC-MS (m / z) 439 (M+1).

[0369] 1 H NMR(400MHz,DMSO-d6)δ10.15(s,1H),8.44(d,J=5.6Hz,1H),8.23(dd,J=10.8, 2.8Hz,1H),7.89(dd,J=10.2,5.6Hz,1H),7.72–7.60(m,3H),7.38(d,J=8.4Hz,2 H),6.46(d,J=5.6Hz,1H),4.05(dd,J=14.0,6.8Hz,1H),3.72–3.51(m,5H),2.74 (d,J=10.0Hz,2H),2.33–2.28(m,2H),2.06–1.92(m,1H),1.24(d,J=4.8Hz,3H).

[0370] Example 55 Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylate

[0371]

[0372] tert-Butyl 6-((6-fluoroquinolin-4-yl)amino)-2-azaspiro[3.3]heptane-2-carboxylate (150 mg, 0.42 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL), and sodium hydroxide (84 mg, 2.1 mmol) was added under nitrogen. The reaction mixture was stirred at room temperature for 5 minutes, followed by the addition of iodomethane (89 mg, 0.63 mmol) under nitrogen. The reaction mixture was heated at 60°C and stirred for 7 hours, then quenched with water. The tetrahydrofuran was removed by distillation under reduced pressure, and the product was extracted with ethyl acetate, washed with water, dried, and concentrated under reduced pressure to obtain a residue. Column chromatography [EA] afforded tert-Butyl 6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylate (50 mg, 32% yield) as a colorless oil. LC-MS (m / z) 372 (M+1).

[0373] Example 56 Preparation of 6-fluoro-N-(2-azaspiro[3.3]heptane-6-yl)quinolin-4-amine hydrochloride

[0374]

[0375] White solid 6-fluoro-N-methyl-N-(2-azaspiro[3.3]heptane-6-yl)quinolin-4-amine hydrochloride (50 mg, 57% yield) was prepared from tert-butyl 6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylate (50 mg, 0.13 mmol) following a procedure similar to that in Example 2. LC-MS (m / z) 272 (M+1).

[0376] Example 57 Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-yl)propanoate

[0377]

[0378] Ethyl 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptan-2-yl)propanoate (50 mg, 83% yield) as a white solid was prepared from 6-fluoro-N-methyl-N-(2-azaspiro[3.3]heptan-6-yl)quinolin-4-amine hydrochloride (50 mg, 0.16 mmol) and ethyl 2-bromopropionate (60 mg, 0.32 mmol) according to a procedure similar to that in Example 37. LC-MS (m / z) 372 (M+1).

[0379] Example 58 Preparation of 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-yl)propanoic acid

[0380]

[0381] White solid 2-(6-((6-Fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]hept-2-yl)propanoic acid (40 mg, 82% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]hept-2-yl)propanoate (50 mg, 0.13 mmol) following a procedure similar to that in Example 38. LC-MS (m / z) 344 (M+1).

[0382] Example 59 Preparation of N-(4-chloro)-2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-yl)propanamide

[0383]

[0384] White solid N-(4-chloro)-2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptan-2-yl)propanamide (6 mg, 11% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)(methyl)amino)-2-azaspiro[3.3]heptan-2-yl)propanoic acid (40 mg, 0.12 mmol) and 4-chloroaniline (20 mg, 0.16 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 453 (M+1).

[0385] 1 H NMR(400MHz, DMSO-d6)δ9.74(s,1H),8.62(d,J=5.2Hz,1H),8.01(dd,J=9.2,5.2Hz,1H),7.75–7.65(m,3H),7.63–7.58(m,1H),7.40–7.32(m,2H ),6.86(d,J=5.2Hz,1H),3.90–3.83(m,1H),3.23(s,2H),3.01(s,1H),2 .84(s,3H),2.16–2.06(m,2H),1.31–1.25(m,4H),1.12(d,J=6.8Hz,3H).

[0386] Example 60 Preparation of tert-butyl 2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridine-5-carboxylate

[0387]

[0388] White solid tert-butyl 2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridine-5-carboxylate (400 mg, 60% yield) was prepared from tert-butyl octahydro-5H-pyrrolo[3,4-c]pyridine-5-carboxylate (457 mg, 1.7 mmol) and 4-chloro-6-fluoroquinoline (350 mg, 1.9 mmol) according to a procedure similar to that in Example 50. LC-MS (m / z) 372 (M+1)

[0389] Example 61 Preparation of 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride

[0390]

[0391] White solid 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride (380 mg, 93% yield) was prepared from tert-butyl 2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridine-5-carboxylate (400 mg, 1.0 mmol) according to a procedure similar to that in Example 2. LC-MS (m / z) 272 (M+1).

[0392] Example 62 Preparation of ethyl 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoate

[0393]

[0394] Ethyl 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoate (380 mg, 73% yield) as a pale yellow oil was prepared from 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride (380 mg, 1.2 mmol) and ethyl 2-bromopropionate (362 mg, 2.4 mmol) according to a procedure similar to that in Example 37. LC-MS (m / z) 372 (M+1).

[0395] Example 63 Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoic acid

[0396]

[0397] White solid 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoic acid (300 mg, 85% yield) was prepared from ethyl 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoate (380 mg, 1.0 mmol) according to a procedure similar to that in Example 38. LC-MS (m / z) 344 (M+1).

[0398] Example 64 Preparation of N-(4-chlorophenyl)-2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanamide

[0399]

[0400] White solid N-(4-chlorophenyl)-2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanamide (20 mg, 15% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoic acid (100 mg, 0.29 mmol) and 4-chloroaniline (74 mg, 0.58 mmol) according to a similar procedure as in Example 39. LC-MS (m / z) 453 (M+1).

[0401] 1 H NMR (400MHz, DMSO-d6) δ9.98 (s, 0.5H), 9.91 (s, 0.5H), 8.38 (dd, J = 5.6, 1.6Hz, 1H), 8.11–8.06 (m, 1H) ,7.87(dd,J=9.2,6.0Hz,1H),7.80–7.73(m,2H),7.58–7.53(m,1H),7.38–7.35(m,2H),6.54(dd,J=5.6 ,3.2Hz,1H),4.11–4.05(m,1H),3.77–3.72(m,1H),3.68–3.60(m,1H),3.50–3.40(m,2H),3.26–3.21( m,1H),2.80–2.67(m,1H),2.62–2.51(m,2H),2.32–2.28(m,2H),1.76–1.61(m,2H),1.23–1.16(m,3H).

[0402] Example 65 Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propionamide

[0403]

[0404] 2-(2-(6-Fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propanamide (15 mg, 12% yield) as a white solid was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoic acid (100 mg, 0.29 mmol) and 3-methoxyaniline (80 mg, 0.60 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 449 (M+1).

[0405] 1H NMR (400MHz, DMSO-d6) δ9.81 (s, 0.5H), 9.76 (s, 0.5H), 8.37 (d, J = 5.6Hz, 1H), 8.10–8.00 (m, 1H), 7 .87(dd,J=9.2,6.0Hz,1H),7.56–7.38(m,2H),7.23–7.20(m,2H),6.66–6.62(m,1H),6.55–6.53(m ,1H),4.03–3.96(m,1H),3.78–3.73(m,3H),3.66–3.60(m,1H),3.52–3.48(m,1H),3.28–3.23(m,1 H),2.78–2.54(m,3H),2.33–2.28(m,2H),2.03–1.97(m,1H),1.74–1.41(m,3H),1.23–1.16(m,3H).

[0406] Example 66 Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(4-methoxyphenyl)propionamide

[0407]

[0408] White solid 2-(2-(6-Fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propanamide (12 mg, 11% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoic acid (85 mg, 0.24 mmol) and 4-methoxyaniline (65 mg, 0.48 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 449 (M+1).

[0409] 1H NMR (400MHz, DMSO-d6) δ9.70 (s, 0.5H), δ9.64 (s, 0.5H), 8.38 (d, J = 5.6Hz, 1H), 8.11–8.06 (m ,1H),7.89–7.85(m,1H),7.63–7.53(m,3H),6.92–6.87(m,2H),6.55–6.53(m,1H),4.08–4.0 4(m,1H),3.78–3.72(m,4H),3.68–3.62(m,1H),3.50–3.46(m,1H),3.23–3.18(m,1H),2.79– 2.67(m,1H),2.615–2.55(m,2H),2.33–2.296(m,2H),1.78–1.61(m,3H),1.23–1.16(m,3H).

[0410] Example 67 Preparation of 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-phenyl-propionamide

[0411]

[0412] White solid 2-(2-(6-Fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)-N-(3-methoxyphenyl)propanamide (10 mg, 10% yield) was prepared from 2-(2-(6-fluoroquinolin-4-yl)octahydro-5H-pyrrolo[3,4-c]pyridin-5-yl)propanoic acid (80 mg, 0.23 mmol) and aniline (45 mg, 0.46 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 419 (M+1).

[0413] 1 H NMR (400MHz, DMSO-d6) δ9.87 (s, 0.5H), δ9.79 (s, 0.5H), 8.42 (d, J = 6.0Hz, 1H), 8.20–8 .15(m,1H),7.93–7.90(m,1H),7.74–7.63(m,3H),7.35–7.29(m,2H),7.09–7.04(m,1H) ,6.62–6.60(m,1H),4.16–4.12(m,1H),3.87–3.72(m,2H),3.61–3.57(m,1H),3.43–3.2 68(m,3H),2.82–2.58(m,3H),2.34–2.29(m,2H),1.76–1.61(m,2H),1.26–1.15(m,3H).

[0414] Example 68 Preparation of tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylate

[0415]

[0416] White solid tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylate (390 mg, 57% yield) was prepared from tert-butyl 6-amino-3-azabicyclo[3.1.0]hexane-3-carboxylate (440 mg, 2.2 mmol) and 4-chloro-6-fluoroquinoline (365 mg, 2.0 mmol) according to a procedure similar to that in Example 50. LC-MS (m / z) 344 (M+1).

[0417] Example 69 Preparation of N-(3-azabicyclo[3.1.0]hexan-6-yl)-6-fluoroquinolin-4-amine hydrochloride

[0418]

[0419] White solid N-(3-azabicyclo[3.1.0]hexan-6-yl)-6-fluoroquinolin-4-amine hydrochloride (300 mg, 95% yield) was prepared from tert-butyl 6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3-carboxylate (390 mg, 1.1 mmol) according to a procedure similar to that in Example 2. LC-MS (m / z) 244 (M+1).

[0420] Example 70 Preparation of ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate

[0421]

[0422] Ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate (350 mg, 90% yield) as a white solid was prepared from 6-fluoro-4-(octahydro-2H-pyrrolo[3,4-c]pyridin-2-yl)quinoline hydrochloride (300 mg, 1.1 mmol) and ethyl 2-bromopropanoate (402 mg, 2.2 mmol) according to a procedure similar to that in Example 37. LC-MS (m / z) 344 (M+1).

[0423] Example 71 Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3-yl)propanoic acid

[0424]

[0425] White solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (320 mg, 95% yield) was prepared from ethyl 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoate (350 mg, 1.0 mmol) according to a procedure similar to that in Example 38. LC-MS (m / z) 316 (M+1).

[0426] Example 72 Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide

[0427]

[0428] 2-(6-((6-Fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propanamide (7 mg, 7% yield) as a white solid was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (80 mg, 0.25 mmol) and 4-chloroaniline (64 mg, 0.5 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 421 (M+1).

[0429] 1 H NMR (400MHz, DMSO-d6) δ9.77(s,1H),8.44(d,J=5.2Hz,1H),7.99(dd,J=11.2,2.8 Hz,1H),7.83(dd,J=9.2,5.2Hz,1H),7.68–7.65(m,2H),7.53–7.48(m,1H),7.39( s,1H),7.36–7.32(m,2H),6.69(d,J=5.2Hz,1H),3.24–3.12(m,3H),2.91(s,1H), 2.73(d,J=8.4Hz,1H),2.65(d,J=8.4Hz,1H),1.68(s,2H),1.24(d,J=6.8Hz,3H).

[0430] Example 73 Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide

[0431]

[0432] White solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-(3-methoxyphenyl)propanamide (12 mg, 11% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (80 mg, 0.25 mmol) and 4-methoxyaniline (65 mg, 0.5 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 421 (M+1).

[0433] 1 H NMR(400MHz,DMSO-d6)δ9.49(s,1H),8.44(d,J=5.2Hz,1H),7.99(dd,J=11.2,2 .8Hz,1H),7.83(dd,J=9.2,5.6Hz,1H),7.54–7.46(m,3H),7.38(s,1H),6.90–6. 82(m,2H),6.69(d,J=5.2Hz,1H),3.70(s,3H),3.21–3.10(m,3H),2.91(s,1H),2 .73(d,J=8.4Hz,1H),2.65(d,J=8.4Hz,1H),1.68(s,2H),1.23(d,J=6.8Hz,3H).

[0434] Example 74 Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-phenyl-propionamide

[0435]

[0436] White solid 2-(6-((6-Fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-phenyl-propionamide (10, 9% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (80 mg, 0.25 mmol) and aniline (60 mg, 0.5 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 391 (M+1).

[0437] 1H NMR (400MHz, DMSO-d6) δ9.63 (s, 1H), 8.45 (d, J = 5.2Hz, 1H), 8.00 (dd, J = 11.2, 2.8Hz, 1H ),7.83(dd,J=9.2,5.6Hz,1H),7.62–7.62(m,2H),7.54–7.49(m,1H),7.42(s,1H),7.28( t,J=7.2Hz,2H),7.04(t,J=7.2Hz,1H),6.70(d,J=5.2Hz,1H),3.23–3.140(m,3H),2.91( s,1H),2.74(d,J=8.4Hz,1H),2.66(d,J=8.4Hz,1H),1.69(s,2H),1.25(d,J=6.8Hz,3H).

[0438] Example 75 Preparation of 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propionamide

[0439]

[0440] White solid 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-N-(4-methoxyphenyl)propanamide (20 mg, 19% yield) was prepared from 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)propanoic acid (80 mg, 0.25 mmol) and 3-methoxyaniline (65 mg, 0.5 mmol) according to a procedure similar to that in Example 39. LC-MS (m / z) 421 (M+1).

[0441] 1 H NMR(400MHz,DMSO-d6)δ9.65(s,1H),8.46(d,J=5.2Hz,1H),8.03(d,J=8.4Hz,1 H),7.86(dd,J=9.2,5.6Hz,1H),7.54(t,1H),7.45(s,1H),7.37(s,1H),7.21–7 .19(m,2H),6.72–6.53(m,2H),3.72(s,3H),3.22–3.19(m,3H),2.91(s,1H),2. 77(d,J=8.4Hz,1H),2.67(d,J=8.4Hz,1H),1.70(s,2H),1.25(d,J=6.8Hz,3H).

[0442] Example 76 Preparation of 2-bromo-3-hydroxypropionic acid

[0443]

[0444] L-serine (500 mg, 4.8 mmol) and sodium bromide were dissolved in 2.5 M aqueous sulfuric acid (10 mL). Sodium nitrite (460 mg, 6.7 mmol) was slowly added under ice-salt bath. The reaction solution was slowly warmed to room temperature and reacted for 15 hours. Water and ethyl acetate were then added for extraction. The mixture was washed with brine and water, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain 2-bromo-3-hydroxypropionic acid (400 mg, 30% yield) as a pale yellow oil. LC-MS (m / z) 169 (M+1)

[0445] Example 77 Preparation of 2-bromo-N-(4-chlorophenyl)-3-hydroxypropionamide

[0446]

[0447] 2-Bromo-3-hydroxypropionic acid (200 mg, 1.2 mmol), p-chloroaniline (100 mg, 0.78 mmol), and N,N-diisopropylethylamine (0.6 mL, 3.1 mmol) were dissolved in tetrahydrofuran (5 mL), followed by the addition of 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (600 mg, 1.6 mmol). The reaction mixture was stirred at room temperature for 5 hours, then poured into water, the pH adjusted to 8-10 with aqueous sodium bicarbonate, extracted with ethyl acetate, washed with water, dried, and concentrated under reduced pressure. The residue was separated by column chromatography (ethyl acetate / petroleum ether = 1 / 2) to obtain 2-bromo-N-(4-chlorophenyl)-3-hydroxypropionamide (100 mg, 46% yield) as a light brown solid. LC-MS (m / z) 279 (M+1).

[0448] Example 78 Preparation of N-(4-chlorophenyl)-2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexan-3-yl)-3-hydroxypropionamide

[0449]

[0450] 2-Bromo-N-(4-chlorophenyl)-3-hydroxypropionamide (65 mg, 0.23 mmol), N-(3-azabicyclo[3.1.0]hexan-6-yl)-6-fluoroquinolin-4-amine hydrochloride (40 mg, 0.16 mmol), and potassium carbonate (68 mg, 48 mmol) were dissolved in acetonitrile (5 mL). The reaction solution was stirred at 80°C for 8 hours and then poured into water. A solid precipitated and was filtered. The residue was separated by column chromatography [methanol / dichloromethane = 1 / 25] to obtain ethyl 2-(6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]hept-2-yl)propanoate (13 mg, 18% yield) as a white solid. LC-MS (m / z) 441 (M+1).

[0451] Example 79 Preparation of tert-butyl 5-((6-fluoroquinolin-4-yl)amino)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate

[0452]

[0453] White solid tert-butyl 5-((6-fluoroquinolin-4-yl)amino)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (200 mg, 65% yield) was prepared from tert-butyl 5-aminohexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (210 mg, 0.9 mmol) and 4-chloro-6-fluoroquinoline (150 mg, 0.82 mmol) according to a procedure similar to that in Example 50. LC-MS (m / z) 372 (M+1).

[0454] Example 80 Preparation of 6-fluoro-N-(octahydrocyclopenta[c]pyrrol-5-yl)quinolin-4-amine hydrochloride

[0455]

[0456] White solid 6-fluoro-N-(octahydrocyclopenta[c]pyrrol-5-yl)quinolin-4-amine hydrochloride (150 mg, 91% yield) was prepared from tert-butyl 5-((6-fluoroquinolin-4-yl)amino)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (200 mg, 0.54 mmol) according to a procedure similar to that in Example 2. LC-MS (m / z) 272 (M+1).

[0457] Example 81 Preparation of ethyl 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)propanoate

[0458]

[0459] White solid 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydropyrrolo[c]pyrrol-2(lH)- yl)propanoic acid ethyl ester (100 mg, 55% yield) was prepared from 6-fluoro-N-( octahydropyrrolo[c]pyrrol-5-yl)quinolin-4-amine hydrochloride (150 mg, 0.49 mmol) and 2-bromoethyl propionate (177 mg, 0.98 mmol) following a similar procedure as in Example 37. LC-MS (m / z) 372 (M+l).

[0460] Example 82 Preparation of 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydropyrrolo[c]pyrrol- 2(lH)-yl)propanoic acid

[0461]

[0462] White solid 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydropyrrolo[c]pyrrol-2(lH)- yl)propanoic acid (80 mg, 87% yield) was prepared from 2-(5-((6-fluoroquinolin-4- yl)amino)hexahydropyrrolo[c]pyrrol-2(lH)-yl)propanoic acid ethyl ester (100 mg, 0.27 mmol) following a similar procedure as in Example 38. LC-MS (m / z) 344 (M+l).

[0463] Example 83 Preparation of N-(4-chlorophenyl)-2-(5-((6-fluoroquinolin-4-yl)amino)hexahydropyrrolo[c]pyrrol-2(lH)-yl)propanamide

[0464]

[0465] Pale yellow solid N-(4-chlorophenyl)-2-(5-((6-fluoroquinolin-4-yl)amino)hexahydropyrrolo[c]pyrrol-2(lH)-yl)propanamide (15 mg, 14% yield) was prepared from 2-(5-((6-fluoroquinolin-4-yl)amino)hexahydropyrrolo[c]pyrrol-2(lH)-yl)propanoic acid (80 mg, 0.23 mmol) and 4-chloroaniline (60 mg, 0.46 mmol) following a similar procedure as in Example 39. LC-MS (m / z) 453 (M+l).

[0466] 1H NMR(400MHz,DMSO-d6)δ9.84(s,1H),8.37(d,J=5.6Hz,1H),8.20(dd,J=11.2,2.8Hz,1 H),7.84(dd,J=9.2,5.6Hz,1H),7.70(d,J=8.8Hz,2H),7.57–7.52(m,1H),7.37(d,J=8. 8Hz,2H),6.93(d,J=6.4Hz,1H),6.56(d,J=5.6Hz,1H),4.24–4.17(m,1H),3.10–3.05( m,1H),2.69–2.66(m,4H),2.44–2.37(m,1H),2.03–1.83(m,5H),1.29(d,J=6.8Hz,3H).

[0467] Example 84 Preparation of Benzo[d][1,3]dioxolan-5-ylglycine Methyl Ester

[0468]

[0469] 3,4-Methylenedioxyaniline (160 mg, 1.16 mmol) was dissolved in 20 ml of DMF, and potassium carbonate (480 mg, 3.48 mmol) and ethyl bromoacetate (213 mg, 1.4 mmol) were added. The mixture was reacted at room temperature for 16 hours. The reaction solution was poured into 40 ml of water and extracted twice with 20 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic phase was spin-dried. The organic phase was separated by column chromatography [ethyl acetate / petroleum ether = 1 / 6] to obtain benzo[d][1,3]dioxolan-5-ylglycine methyl ester (175 mg, 72% yield) as a brown oil. LC-MS (m / z) 210 (M+H).

[0470] Example 85 Preparation of Benzo[d][1,3]dioxolan-5-ylglycine

[0471]

[0472] Benzo[d][1,3]dioxolan-5-ylglycine methyl ester (175 mg, 3.42 mmol) was dissolved in 20 ml of methanol, and sodium hydroxide (164 mg, 4.1 mmol) was added. The mixture was reacted at room temperature for 16 hours, poured into 20 ml of water, and the pH was adjusted to 4 with hydrochloric acid. The mixture was then extracted twice with 20 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic phase was dried to give brown solid benzo[d][1,3]dioxolan-5-ylglycine (160 mg, 98% yield). LC-MS (m / z) 196 (M+H).

[0473] Example 86 Preparation of 2-(Benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl)piperidin-1-yl)ethyl-1-one

[0474]

[0475] 1-Boc-4-[2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl]piperidine (100 mg, 0.25 mmol) was dissolved in 20 ml of dichloromethane, and 0.5 ml of hydrochloric acid was added. The mixture was reacted at room temperature for 2 hours, dried by rotation, and 10 ml of DMF was added and stirred until it was completely dissolved. Benzo[d][1,3]dioxolan-5-ylglycine (58.5 mg, 0.3 mmol) and 1-hydroxybenzotriazole (67.7 mg, 0.5 mmol) were added, along with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (95.6 mg, 0.5 mmol). mol), 1 ml of N,N-diisopropylethylamine, reacted at room temperature for 16 hours, poured into 40 ml of water, extracted twice with 30 ml of ethyl acetate, combined the organic phases, dried over anhydrous sodium sulfate, filtered, and the organic phase was spin-dried. The product was separated by column chromatography [dichloromethane / methanol = 100 / 3] to give 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl)piperidin-1-yl)ethyl-1-one (50 mg, 42% yield) as a white solid, LC-MS (m / z) 477 (M+H).

[0476] Example 87 Preparation of 2-(Benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one

[0477]

[0478] 2-(Benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)acetyl)piperidin-1-yl)ethyl-1-one (50 mg, 0.1 mmol) was dissolved in 10 ml of methanol. Under nitrogen protection, the temperature was cooled to 0 degrees with an ice bath. Sodium borohydride (11.3 mg, 0.1 mmol) was added and reacted at room temperature for 2 hours. 1 ml of saturated aqueous ammonium chloride solution was added and stirred for 5 minutes. , poured into 20 ml of water, extracted twice with 20 ml of ethyl acetate, combined the organic phases, dried over anhydrous sodium sulfate, spin-dried the organic phase, and separated by column chromatography (dichloromethane / methanol = 20 / 1) to obtain a light brown solid 2-(benzo[d][1,3]dioxolan-5-ylamino)-1-(4-(2-(6-fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)piperidin-1-yl)ethyl-1-one (15 mg, 30% yield).

[0479] 1 H NMR(400MHz,DMSO-d6)δ8.00(s,0.7H),7.97(s,0.3H),7.47-7.42(m,2H),7.22(s,0.3H),7.20(s,0.7H),7.14-7.07(m,1H ),6.65(d,J=8.4Hz,1H),6.42(d,J=2.0Hz,1H),6.07(dd,J=8.4,2.0Hz,1H),5.83(s,2H),5.72-5.67(m,0.3H),5.64-5.57( m,0.7H),5.19-5.11(m,0.3H),4.73(t,J=4.8Hz,0.8H),4.42(t,J=12.5Hz,1H),3.94(s,br,1H),3.80(s,2H),3.43(s,br,1 H),3.00-2.82(m,1H),2.41-2.23(m,1H),2.04-1.86(m,0.7H),1.87-1.76(m,0.3H),1.74-1.42(m,3H),1.37-0.97(m,4H).

[0480] LC-MS (m / z) 479 (M+H).

[0481] In vitro biological evaluation

[0482] The purpose of this assay is to comprehensively evaluate the in vitro and cell model inhibitory activities of different compounds against human indoleamine 2,3-dioxygenase 1 (hIDO1).

[0483] Example 88 Cellular Level Inhibitory Activity Detection

[0484] In addition to being constitutively expressed in immune cells such as myeloid-derived suppressor cells (MDSCs), IDO1 is also upregulated in many tumor cells or induced by cytokines such as IFN-γ. In this application, the inventors used IFN-γ-induced IDO1 expression in Hela cells as a model to test the inhibitory activity of compounds on the IDO1 enzyme at the cellular level.

[0485] Main experimental principles

[0486] HeLa cells are a human cervical cancer cell line that upregulates endogenous IDO1 expression in response to human IFN-γ. Adding the substrate L-tryptophan to the cell culture medium allows for the detection of kynurenine, the enzyme's catalytic product, in the cell supernatant. This assay utilizes cultured HeLa cells stimulated with human IFN-γ and incubated with various concentrations of test compounds for designated periods of time. The effect of test compound treatment on cellular IDO enzyme activity is assessed using a colorimetric reaction between the enzyme product and p-dimethylaminobenzaldehyde.

[0487] Experimental materials and reagents

[0488] Recombinant human IFN-γ cytokine was purchased from Sangon Biotechnology Co., Ltd., and phenol red-free DMEM for cell culture was purchased from Gibco. Detection reagents, including L-tryptophan (Sangon, A601911-0050), kynurenine (Sigma, K8625-100MG), trichloroacetic acid (Sangon, A600968-0250), and p-dimethylaminobenzaldehyde (Tianjin Damao Chemical Reagent Factory), were also purchased from Corning, Costar 3599. Cell culture 96-well flat-bottom plates were also used.

[0489] Experimental methods

[0490] The cells were cultured in a 96-well flat-bottom plate according to conventional cell culture experimental procedures.

[0491] (1) HeLa cells were seeded into 96-well culture plates at an appropriate concentration (approximately 20,000 cells / well). After overnight adherence, the culture medium was changed to phenol red-free DMEM containing 200 μM L-tryptophan. 50 ng / ml of human IFN-γ and different concentrations of the test compound (maximum final concentration 25 μM) and NLG919 were added. A solvent control (DMSO) and negative control wells without cytokines and L-tryptophan were also set up in triplicate. The cells were cultured for 48 hours before testing.

[0492] (2) 200 μL of the supernatant in the culture well was aspirated, 40 μL of pre-prepared 30% (w / v) trichloroacetic acid solution was added, and the mixture was reacted at 65°C for 20 min, followed by centrifugation at 12,000 rpm for 15 min.

[0493] (3) 100 μL of the supernatant after centrifugation was added to a 96-well flat-bottom plate, and an equal volume of 2% p-dimethylaminobenzaldehyde solution in glacial acetic acid was added, mixed, and allowed to stand at room temperature for 10 min.

[0494] (4) Use an enzyme-labeled instrument (ELX800NB) to detect the absorbance value of each well at a detection wavelength of 492 nm.

[0495] (5) Calculation formula for the cellular enzyme inhibition rate of the test compound:

[0496] Enzyme activity inhibition rate (%) = (OD 溶剂对照 -OD 化合物检测孔 ) / (OD 溶剂对照 -OD 阴性对照 )*100%.

[0497] In addition, the cell enzyme activity inhibition rate was calculated for each test compound at different concentration gradients, and the half-inhibitory concentration (EC50) of the cell enzyme activity was calculated using the EC50 calculator. 50 ).

[0498] According to the above experimental method, the compounds described in this application were subjected to cellular level IDO1 enzymatic evaluation (the test compound concentration was 100 nM). The data summary is shown in Table 1.

[0499] Table 1 Data on hIDO1 cell level inhibition rate of representative compounds in this application

[0500]

[0501]

[0502] According to the above experimental method, the compounds described in this application were subjected to EC 50 The data are summarized in Table 2.

[0503] Table 2 EC levels of hIDO1 cells of representative compounds of this application 50 (nM)

[0504]

[0505]

[0506] Example 89 Experiment on the inhibition of IDO enzyme activity in HeLa cells by combining Chidamide with the compound of Example 3

[0507] Experimental Materials

[0508] Human cervical cancer cell lines (HeLa) were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured at 37°C in 5% CO2. The culture medium was DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (HyClone). Trypsin was purchased from Gibco. Recombinant human IFN-γ cytokine was purchased from Sangon Biotechnology Co., Ltd. Phenol red-free DMEM for cell culture was purchased from Gibco. Detection reagents included L-tryptophan (Sangon, A601911-0050), kynurenine (Sigma, K8625-100MG), trichloroacetic acid (Sangon, A600968-0250), and p-dimethylaminobenzaldehyde (Tianjin Damao Chemical Reagent Factory). Cell culture was performed in 96-well flat-bottom plates (CORNING, Costar 3599).

[0509] Experimental methods

[0510] HeLa cells were collected by trypsin digestion and counted, with a concentration of 2 × 10 cells per well. 4 Cells were seeded into 96-well cell culture plates and cultured at 37°C with 5% CO2. After the cells were seeded overnight, the culture medium was changed to phenol red-free DMEM medium containing 200 μM L-tryptophan, and 50 ng / ml of cytokine human IFN-γ and different concentrations of drugs (according to the Figure 1 The cells were divided into groups and administered with final concentrations as shown in the table), and solvent controls (DMSO) and negative control wells without cytokines and L-tryptophan were set up at the same time, and the wells were set up in triplicate. After 48 hours of drug treatment, 200 μL of the supernatant in the culture wells was aspirated, 40 μL of a pre-prepared 30% (w / v) trichloroacetic acid solution was added, and the cells were reacted at 65°C for 20 minutes. Subsequently, the cells were centrifuged at 12,000 rpm for 15 minutes. 100 μL of the supernatant after centrifugation was aspirated and added to a 96-well flat-bottom plate, and an equal volume of 2% p-dimethylaminobenzaldehyde glacial acetic acid solution was added and mixed, and the cells were placed at room temperature for 10 minutes. The absorbance value at a wavelength of 492 nm for each well was read by an enzyme reader (ELX800NB). After subtracting the OD492-BLK of the cell-free culture medium as a blank control from the readings of each well, the OD492-T of each dosing well and the OD490-T0 of the drug-free positive control well were obtained after subtracting the detection background from the readings of each well.

[0511] The relative IDO enzyme activity inhibition rate of cells in each dosing well was calculated according to the following formula:

[0512] IDO enzyme activity inhibition rate = (OD 492-T0 -OD 492-T ) / OD 492-T0 ×100%

[0513] Experimental results

[0514] like Figure 1 As shown in Table 3, compared with the solvent control, both chidamide and the compound of Example 3 showed a certain inhibitory effect on IDO enzyme activity; and the combination of the two drugs showed a significant synergistic or additive inhibitory effect. Considering that HDAC inhibitors do not directly bind to and inhibit the catalytic activity of IDO protease, but indirectly inhibit IDO activity by downregulating IDO mRNA to reduce its protein expression (He YW, et al. Life Sci, 2013, 93(15): 509-515), the combination of epigenetic inhibitors and IDO inhibitors utilizes different mechanisms to exert a synergistic IDO enzyme inhibitory effect.

[0515] Table 3 IDO enzyme activity inhibition rate of chidamide and the compound of Example 3 alone or in combination

[0516] Solvent control Chidamide Example 3 Two-drug combination Average inhibition rate (%) 0.5 24 31 49

[0517] Example 90 Experiment on the inhibition of IDO enzyme activity in HeLa cells by combining Chidamide with Example 20

[0518] A similar method as in Example 89 was used. The experimental results obtained are as follows Figure 2 and as shown in Table 4.

[0519] Table 4 IDO enzyme activity inhibition rate of chidamide and the compound of Example 20 alone or in combination

[0520] Solvent control Chidamide Example 20 Two-drug combination Average inhibition rate (%) 0.5 22 16 35

[0521] Example 91 Experiment on the inhibition of IDO enzyme activity in HeLa cells by combining Chidamide with Example 72

[0522] A similar method as in Example 89 was used. The experimental results obtained are as follows Figure 3 and as shown in Table 5.

[0523] Table 5 IDO enzyme activity inhibition rate of chidamide and the compound of Example 72 alone or in combination

[0524] Solvent control Chidamide Example 72 Two-drug combination Average inhibition rate (%) 0.5 23 36 58

[0525] Example 92 Experiment on the inhibition of IDO enzyme activity in HeLa cells by combining Chidamide with Example 87

[0526] A similar method as in Example 89 was used. The experimental results obtained are as follows Figure 4 and as shown in Table 6.

[0527] Table 6 IDO enzyme activity inhibition rate of chidamide and the compound of Example 87 alone or in combination

[0528] Solvent control Chidamide Example 87 Two-drug combination Average inhibition rate (%) 0.5 23 20 47

[0529] Example 93 Pharmacodynamic experiment of chidamide combined with Example 3 in Balb / c mouse CT-26 tumor model

[0530] Experimental materials

[0531] The mouse colon cancer cell line CT-26 was purchased from the Cell Resource Center of Shanghai Life Sciences Institute, Chinese Academy of Sciences, and was routinely cultured at 37°C in a 5% CO2 environment. The culture medium was DMEM (Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% Penicillin-Streptomycin (HyClone). Trypsin was purchased from Gibco. Normal Balb / c mice were purchased from the Guangdong Medical Experimental Animal Center.

[0532] Experimental method

[0533] A large number of CT-26 cells were cultured and kept in a logarithmic growth state. After the cell number reached the required amount, the cells were collected by trypsin digestion, washed with a large amount of PBS for 2 times to remove trypsin and serum components, centrifuged at 800 rpm for 10 min at room temperature, and the supernatant was discarded. The cells were resuspended with FBS-free DMEM culture medium, and the cell concentration was adjusted to 3x10 7 / mL.

[0534] Under sterile conditions, 100 μL / needle of the cell suspension was injected subcutaneously into the back of the nude mice, and each Balb / c mouse was injected with one needle. A 1 mL disposable medical syringe was used for injection to ensure that the injection site and direction of each mouse were basically consistent.

[0535] After 8 days of cell inoculation, the tumors grew to an average volume of about 100 mm 3Tumor-bearing mice were randomly divided into four groups (nine mice per group): a solvent control group, a chidamide group (25 mg / kg, gavage, once daily), a group receiving the compound of Example 3 (20 mg / kg, gavage, once daily), and a group receiving a combination of chidamide and the compound of Example 3. After labeling, the mice were housed in separate cages and dosed daily according to group, and tumor formation was observed. Each mouse was weighed before dosing, and the dosage was calculated per kilogram of body weight. The solvent control group received 10 μL of CMC-Na solution per gram of body weight, the chidamide group (25 mg / kg) received 10 μL of a 2.5 mg / mL chidamide-CMC-Na suspension per gram of body weight, the Example 3 group (20 mg / kg) received 10 μL of a 2 mg / mL Example 3-CMC-Na suspension per gram of body weight, and the combination group received 10 μL of a CMC-Na suspension containing 2.5 mg chidamide and 2 mg Example 3 per milliliter per gram of body weight. Every 2 days, the longest diameter (length) and the widest diameter (width) perpendicular to the tumor were measured with a vernier caliper. The formula TS = length × (width) was used. 2 The tumor volume was calculated and recorded. Each mouse was given the drug once daily by oral gavage. After the last administration on day 17, the mice were sacrificed and the tumors of the tumor-bearing mice were removed and weighed using a scale.

[0536] The relative tumor inhibition rate of each drug-treated group was calculated according to the following formula:

[0537] Relative tumor inhibition rate = (average tumor volume of solvent control group - average tumor volume of drug administration group) / average tumor volume of solvent control group × 100%

[0538] Experimental results

[0539] like Figure 5 As shown in Table 7, compared with the solvent control group, the two groups of single-dose chidamide (25 mg / kg) and the compound of Example 3 (20 mg / kg) had a certain inhibition on the tumor volume of mice, with tumor inhibition rates of 33% and 20%, respectively; and the final relative tumor inhibition rate of the combination group (80%) was significantly higher than the sum of the tumor inhibition rates of the two single-drug groups. Figure 6 As shown, compared to the average tumor weight of 2.7g in the solvent group, the average tumor weights of the chidamide and Example 3 single-agent groups were 1.6g and 2.3g, respectively, showing some inhibition. The average tumor weight in the combination group was only 0.35g, significantly less than the two single-agent groups. These results demonstrate that chidamide and IDO inhibitors have good synergistic antitumor activity in tumor-bearing mice.

[0540] Table 7 Relative tumor inhibition rates of chidamide and the compound of Example 3 alone or in combination

[0541] Chidamide Example 3 Two-drug combination group Relative tumor inhibition rate (%) 33.6 20.1 80.8

[0542] Example 94: Efficacy Experiment of Chidamide Combined with Example 20 in Balb / c Mouse CT-26 Tumor Model

[0543] A similar method as in Example 93 was used. The experimental results obtained are as follows Figure 7 and shown in Table 8.

[0544] Table 8 Relative tumor inhibition rates of chidamide and the compound of Example 3 alone or in combination

[0545] Chidamide Example 20 Two-drug combination group Relative tumor inhibition rate (%) 30 8.3 87.7

[0546] Example 95: Efficacy experiment of chidamide combined with Example 72 in a Balb / c mouse CT-26 tumor model

[0547] A similar method as in Example 93 was used. The experimental results obtained are as follows Figure 8 and as shown in Table 9.

[0548] Table 9 Relative tumor inhibition rates of chidamide and the compound of Example 72 alone and in combination

[0549] Chidamide Example 72 Two-drug combination group Relative tumor inhibition rate (%) 25.4 7.6 86.9

[0550] Example 96: Efficacy experiment of chidamide combined with Example 87 in Balb / c mouse CT-26 tumor model

[0551] A similar method as in Example 93 was used. The experimental results obtained are as follows Figure 9 and shown in Table 10.

[0552] Table 10 Relative tumor inhibition rates of chidamide and the compound of Example 87 alone or in combination

[0553] Chidamide Example 87 Two-drug combination group Relative tumor inhibition rate (%) 55 45 89.8

[0554] Example 97 hERG inhibitory activity detection (patch clamp method)

[0555] Non-cardiac drugs may prolong myocardial action potential duration by inhibiting hERG (IKr) channels, increasing the likelihood of life-threatening ventricular arrhythmias such as torsade de pointes (TdP). This study used the HEK293 cell line, which lacks an endogenous IKr current and is widely used for hERG detection, as the host cell to evaluate the cardiotoxicity of Example 85 in CN108203438A and Example 87 in this application.

[0556] 1. Cell culture

[0557] (1) HEK293 cells stably expressing hERG potassium channel were cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg / mL G418 at a temperature of 37° C. and a carbon dioxide concentration of 5%.

[0558] (2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of TrypLETM Express solution and incubate at 37°C for 0.5 min. When the cells detach from the bottom of the dish, add 5 mL of complete culture medium preheated at 37°C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min to collect the cells. For expansion or maintenance culture, inoculate the cells into a 6 cm cell culture dish. The inoculated cell amount for each cell culture dish is 2.5*105 cells (final volume: 5 mL).

[0559] (3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0560] (4) Patch clamp assay: Before the experiment, cells were stained with TrypLE TM Express separation, 3*103 cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). Experimental detection was performed after 18 hours.

[0561] 2. Liquid configuration

[0562] The composition of the extracellular solution used in the whole-cell patch-clamp experiment was (mM): NaCl 140; MgCl2 1; KCl 3.5; Glucose 10; HEPES 10; NaH2PO4 1.25 and CaCl2 2. The pH was adjusted to 7.4 with NaOH, and the osmotic pressure was adjusted to 300 mOsm with sucrose.

[0563] The components of the intracellular solution were (mM): KCl 20; K-Aspartic acid 115; MgCl2 1; EGTA 5; HEPES 10; and Na2ATP 2. The pH was adjusted to 7.2 with KOH, and the osmotic pressure was adjusted to 290 mOsm with sucrose.

[0564] 3. Patch clamp detection

[0565] The voltage stimulation protocol for whole-cell patch clamp recording of whole-cell hERG potassium current is as follows: after the whole-cell seal is formed, the cell membrane voltage is clamped at -80mV. The clamping voltage is depolarized from -80mV to -50mV for 0.5s (as a leakage current detection), then stepped to 30mV for 2.5s, and then quickly restored to -50mV for 4s to stimulate the tail current of the hERG channel. Data is collected repeatedly every 10s to observe the effect of drugs on the hERG tail current. A 0.5s stimulation of -50mV is used for leakage current detection. The experimental data are collected by an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0566] Recording electrodes are drawn from capillary glass tubes using a microelectrode puller. Under an inverted microscope, the microelectrode manipulator is used to place the recording electrode in contact with the cell. Negative pressure is applied to create a GΩ seal. After the GΩ seal is established, rapid capacitance compensation is performed. Continued negative pressure is applied to rupture the cell membrane, establishing whole-cell recording mode. Slow capacitance compensation is then performed, and membrane capacitance and series resistance are recorded. No leakage compensation is applied.

[0567] When the hERG current recorded in the whole cell is stable, the drug is administered. After each drug concentration is applied for 5 minutes (or the current is stable), the next concentration is detected. Multiple concentrations are detected for each test product. The coverslip with cells is placed in the recording chamber of an inverted microscope. The test sample working solution and the external solution without compounds are flowed through the recording chamber from low concentration to high concentration by gravity perfusion to act on the cells. A vacuum pump is used for liquid exchange during recording. The current detected in the external solution without compounds for each cell serves as its own control group. Multiple cells are tested independently and repeatedly. All electrophysiological experiments are performed at room temperature.

[0568] Drug concentrations were selected as 0.3, 1, 3, 10, and 30 μM.

[0569] 4. Data Analysis

[0570] First, the current after each concentration was normalized with the blank control current. Then calculate the inhibition rate corresponding to each concentration The mean and standard error were calculated for each concentration, and the half-inhibitory concentration (CI) of each compound was calculated using the following equation:

[0571]

[0572] The dose-dependent effects were fitted nonlinearly using the above equation, where C represents the test article concentration, IC50 is the half-inhibitory concentration, and h represents the Hill coefficient. Curve fitting and IC50 calculation were performed using IGOR software.

[0573] According to the above experimental method, the hERG inhibition EC50 value of Example 85 in patent CN108203438A was determined to be 1.9 uM. The hERG inhibition EC50 value of Example 87 of this patent was 4.6 uM.

[0574] Although the specific embodiments of the present invention have been described in detail, based on all the teachings disclosed, those skilled in the art may make various modifications and substitutions to the details of the technical solution of the present invention, and such modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, AXBYM (I) in, A represents quinolyl and 6-fluoroquinolyl; X represents -O-, -NH- or -N(CH3)-; B is selected from Y represents -C(O)-, M represents a benzene ring or a benzo[1,3]dioxole ring; optionally, the benzene ring is substituted by a substituent selected from the group consisting of fluorine, chlorine, methoxy and methoxy-substituted propenyl.

2. The compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein A represents 6-fluoroquinolyl; X stands for -O-; B is selected from Y stands for M represents a benzene ring or a benzo[1,3]dioxole ring; the benzene ring is substituted by a substituent selected from the group consisting of fluorine and chlorine.

3. A pharmaceutical composition comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition of claim 3, wherein the compound is selected from N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide, N-(benzo[d][1,3]dioxolan-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide, 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3-yl)-N-(4-methoxyphenyl)propionamide; and further contains cedamide.

5. A combination drug comprising a first active ingredient and a second active ingredient, and optionally a pharmaceutically acceptable carrier or excipient; wherein: The first active ingredient is selected from N-(4-chlorophenyl)-6-((6-fluoroquinolin-4-yl)oxy)-2-aza-spiro[3.3]heptane-2-carboxamide, N-(benzo[d][1,3]dioxolane-5-yl)-6-((6-fluoroquinolin-4-yl)oxy)-2-azaspiro[3.3]heptane-2-carboxamide, and 2-(6-((6-fluoroquinolin-4-yl)amino)-3-azabicyclo[3.1.0]hexane-3-yl)-N-(4-methoxyphenyl)propionamide; the second active ingredient is selected from cedamide. The combined drug according to claim 5 , wherein the first active ingredient and the second active ingredient are in the same preparation unit.

7. The combination drug according to claim 5, wherein the first active ingredient and the second active ingredient are in different preparation units.

8. The combined drug according to claim 5, wherein the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially.

9. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, the pharmaceutical composition according to claim 3 or 4, or the combination drug according to any one of claims 5 to 8 in the preparation of an immunomodulator or a medicament for preventing and / or treating diseases associated with abnormal IDO expression and / or abnormal tryptophan metabolism.

10. The use according to claim 9, wherein the disease is selected from tumors, autoimmune diseases, cataracts, Alzheimer's disease, depressive disorders and anxiety disorders.

Images